N-terminus conformationally constrained glp-1 receptor agonist compounds

ABSTRACT

The disclosure provides N-terminus conformationally constrained compounds, which may comprise peptide mimetics and/or amino acid substitutions, which may be used in peptides, such as GLP-1 receptor agonist compounds, to induce a β-turn secondary structure at the N-terminus. The N-terminus conformationally constrained compounds may be used for research purposes; to produce GLP-1 receptor agonist compounds having improved GLP-1 receptor binding activity, enzymatic stability, or in vivo glucose lowering activity; and to develop GLP-1 receptor agonist compounds which have fewer amino acid residues. The disclosure also provides GLP-1 receptor agonist compounds, such as exendins, exendin analogs, GLP-1 (7-37), GLP-1 (7-37) analogs, comprising the N-terminus conformationally constrained compounds. The compounds are useful for treating various diseases, such as diabetes and obesity. The disclosure also provides methods for chemically synthesizing the N-terminus conformationally constrained compounds.

RELATED APPLICATIONS

This application claims priority to U.S. Application No. 61/165,604filed Apr. 1, 2009.

FIELD

Provided herein are N-terminus conformationally constrained GLP-1receptor agonist compounds and therapeutic methods for their use.

BACKGROUND

Peptides and proteins play critical roles in the regulation ofbiological processes. Peptides, for example, play a regulatory role ashormones and inhibitors, and are also involved in immunologicalrecognition. The significant biological role of peptides makes itimportant to understand their interactions with the receptors to whichthey bind.

The determination of the receptor-bound conformation of a peptide isinvaluable for the rational design of peptide analogs. Marshall et al,Ann. Rep. Med. Chem., 13:227-238 (1978) disclose that peptides arecharacteristically highly flexible molecules, the structures of whichare strongly influenced by the environment in which they reside. Thus,peptides are not generally useful for determining their receptor-boundconformation.

As no approach is available to predict which new ligand-receptorinteractions will lead to antagonists and which will lead to agonists ofgreater or less potency, it is necessary to perform classicalstructure-function studies in a systematic way to provide informationabout the specific amino acid residues and functional groups in apeptide that are important to biological activity. Studies of thisnature can utilize conformationally constrained peptide mimetics. Forexample, Hruby, Trends Pharmacol. Sci., 8:336-339 (1987) suggests thatconformational constraints can provide information about the differentrequirements that a receptor has for a ligand to be an agonist orantagonist.

Generally, peptide mimetics can be defined as structures which serve asappropriate substitutes for peptides in interactions with receptors andenzymes. The development of rational approaches for discovering peptidemimetics is a major goal of medicinal chemistry. Such development hasbeen attempted both by empirical screening approaches and by specificsynthetic design. Specific design of peptide mimetics has utilized bothpeptide backbone modifications and chemical mimics of peptide secondarystructures. The beta-turn has been implicated as an important site formolecular recognition in many biologically active peptides.Consequently, peptides containing conformationally constrained mimeticsof beta-turns are particularly desirable.

There is a need in the art for new GLP-1 receptor agonist compounds thathave good stability, resistance to degradation, and good glucagon-likepeptide-1 (GLP-1) receptor binding activity and in vivo glucose loweringactivity. To solve these needs, the disclosure herein provides, amongother things, novel N-terminus conformationally constrained compounds,novel N-terminus conformationally constrained GLP-1 receptor agonistcompounds containing modifications, such as peptide mimetics and/oramino acid substitutions, that provide a conformationally constrainedN-terminus that results in improved GLP-1 receptor binding and in vivoblood glucose lowering activity.

SUMMARY

It was previously believed that the N-terminus of exendin-4 and exendinanalogs was a random coil. It has now been unexpectedly discovered thatthe N-terminus shows a high beta-turn characteristic in a specific site,and therefore mimics the receptor bound conformation of this region ofthe peptides. The disclosure herein is based on this discovery.

Provided herein are N-terminus conformationally constrained compoundshaving the formula: Xaa₁Xaa₂Xaa₃-Z and Xaa₁Xaa₂Xaa₃Xaa₄-Z, where thesubstituents are defined herein. These N-terminus conformationallyconstrained compounds may induce a β-turn conformational constraint atthe N-terminus when they are used in GLP-1 receptor agonist compounds.The N-terminus conformationally constrained compounds may be used fortherapeutic purposes (e.g., treat diabetes); for research purposes; andto produce GLP-1 receptor agonist compounds having improved GLP-1receptor binding activity, enzymatic stability, and improved in vivoglucose lowering activity. The disclosure provides pharmaceuticalcompositions comprising therapeutically effective amounts of theN-terminus conformationally constrained compounds. The disclosure alsoprovides methods for synthesizing the N-terminus conformationallyconstrained compounds.

Provided herein are GLP-1 receptor agonist compounds, such as exendins,exendin analogs, GLP-1(7-37), and GLP-1(7-37) analogs, comprising anN-terminus conformationally constrained compound having the formulaXaa₁Xaa₂Xaa₃- or Xaa₁Xaa₂Xaa₃Xaa₄-, where the substituents are definedherein. In one embodiment, the GLP-1 receptor agonist compounds compriseXaa₁Xaa₂Xaa₃Xaa₄, where the substituents are defined herein, atpositions 1-4 at the N-terminus. In one embodiment, the GLP-1 receptoragonist compounds comprise Xaa₁Xaa₂Xaa₃, where the substituents aredefined herein, at positions 1-3 at the N-terminus. The disclosureprovides pharmaceutical compositions comprising therapeuticallyeffective amounts of these N-terminus conformationally-constrained GLP-1receptor agonist compounds.

Provided herein are exendins and exendin analogs having the formula:

Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKN-Z;

Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LK-R₁₀-Z;

Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKNGGPSSGAPPPS-Z;

where Xaa₁₄, Xaa₂₅, R₁₀, and Z are defined herein; and at least one ofXaa₁, Xaa₂, Xaa₃, and Xaa₄ are modifications, such as peptide mimeticsand/or amino acid substitutions, that induce a conformational constraintat the N-terminus. The disclosure provides pharmaceutical compositionscomprising therapeutically effective amounts of these exendin analogs.

Provided herein are GLP-1(7-37) and GLP-1(7-37) analogs having theformula:

Xaa₁Xaa₂Xaa₃Xaa₄TFTSDVSSYXaa₁₄EGQAAKEFIAXaa₂₅LVXaa₂₈GRXaa₃₁-Z;

where Xaa₁₄, Xaa₂₅, Xaa₂₈, Xaa₃₁, and Z are as defined herein; and atleast one of Xaa₁, Xaa₂, Xaa₃, and Xaa₄ are modifications, such aspeptide mimetics and/or amino acid substitutions, that induce aconformational constraint at the N-terminus. The disclosure providespharmaceutical compositions comprising therapeutically effective amountsof these GLP-1(7-37) analogs.

Provided herein are GLP-1 receptor agonist compounds, such as exendins,exendin analogs, GLP-1(7-37), and GLP-1(7-37) analogs wherein position 1comprises an imidazole ring (e.g., His) and position 3 is proline; wherethe GLP-1 receptor agonist compounds bind in a RIN cell membranereceptor binding assay with an affinity of less than 1 nM (or less than0.1 nM).

The disclosure provides methods for treating diabetes; treating insulinresistance; treating postprandial hyperglycemia; lowering blood glucoselevels; lowering HbA1c levels; stimulating insulin release; reducinggastric motility; delaying gastric emptying; reducing food intake;reducing appetite; reducing weight; treating overweight; and treatingobesity in patients in need by administering therapeutically effectiveamounts of the N-terminus conformationally constrained compounds and/orthe N-terminus conformationally constrained GLP-1 receptor agonistcompounds described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: FIG. 1A is exendin-4 amide; FIG. 1B is dAla²,Pro³-exendin-4amide, and FIG. 1C is dAla²,Pro³,Leu¹⁴,Phe²⁵-exendin-4 (1-28) amide.FIG. 1D is a graph showing the change in blood glucose in miceadministered the compounds shown in FIGS. 1A-C based on the in vivoblood glucose assay described in Example 17. The compounds were injectedIP at t=0 immediately following a baseline sample in 2-hour fastedNIH/Swiss mice. Blood glucose samples were taken at t=30, 60, 120, 180,and 240 minutes with a ONETOUCH® ULTRA® (LifeScan, Inc., Milpitas,Calif.).

FIG. 2: FIG. 2A is an exendin analog, described, e.g., in WO2007/139941. FIGS. 2B-J show the exendin analog of FIG. 2A having amodification at Glu³. R₁ is GTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS-NH₂. FIG.2K shows the exendin analog of FIG. 2A having modifications at Gly²Glu³.FIGS. 2L-2U show N-terminus conformationally constrained compounds.FIGS. 2B-J provide examples of exendin analogs containing the N-terminusconformationally constrained compounds shown in FIGS. 2L-2U.

FIGS. 3A-G are dAla²,Pro³-exendin-4 (FIG. 3A), Ala²,Pro³-exendin-4 (FIG.3B), Pro³,Ala⁴-exendin-4 (FIG. 3C), Ala²,Pro³,Ala⁴-exendin-4 (FIG. 3D),Pro³,dAla⁴-exendin-4 (FIG. 3E), Val²,Pro³-exendin-4 (FIG. 3F), andNMeAla²,Pro³-exendin-4 (FIG. 3G). The compound in FIGS. 1B and 3A arethe same.

FIG. 4 is a graph showing the change in blood glucose in miceadministered the compounds shown in FIGS. 3G, 2I, and 2B based on the invivo blood glucose assay described in Example 17. The compounds wereinjected IP at t=0 immediately following a baseline sample in 2-hourfasted NIH/Swiss mice. Blood glucose samples were taken at t=30, 60,120, 180, and 240 minutes with a ONETOUCH® ULTRA® (LifeScan, Inc.,Milpitas, Calif.).

FIG. 5: FIG. 5A is Leu¹⁴,Phe²⁵-exendin-4(1-28), described in WO2007/139941. Leu¹⁴,Phe²⁵-exendin-4(1-28) refers to amino acid residues1-28 in exendin-4 (i.e., exendin-4(1-28), where the amino acid residueat position 14 in exendin-4 is replaced with Leu (i.e., Leu¹⁴), and theamino acid residue at position 25 is replaced with Phe (i.e., Phe²⁵).FIGS. 5B-G show the exendin analog of FIG. 5A having a modification atGlu³ or Gly²Glu³ at the N-terminus. R₄ is GTFTSDLSKQLEEEAVRLFIEFLKN-NH₂.

FIGS. 6A-E show the exendin analog of FIG. 5A having a modification atGly²Glu³ or Gly²Glu³Gly⁴ at the N-terminus. The C-terminal amino acid ineach compound shown in FIGS. 6A-E is amidated.

FIG. 7 is a graph showing the change in blood glucose in miceadministered the compounds shown in FIGS. 6A, 8B, 6E, 1C, 5B, and 5Abased on the in vivo blood glucose assay described in Example 17. Thecompounds were injected IP at t=0 immediately following a baselinesample in 2-hour fasted NIH/Swiss mice. Blood glucose samples were takenat t=30, 60, 120, 180, and 240 minutes with a ONETOUCH® ULTRA®(LifeScan, Inc., Milpitas, Calif.).

FIG. 8: FIGS. 8A-D show the exendin analog in FIG. 5A havingmodifications at Gly²Glu³Gly⁴ at the N-terminus, where the C-terminalamino acid is amidated. FIGS. 8E-H show the exendin analog in FIG. 2Ahaving modifications at Gly²Glu³Gly⁴ at the N-terminus.

FIG. 9: FIGS. 9A-H show the exendin analog in FIG. 5A having amodification at Gly²Glu³ or Gly²Glu³Gly⁴ at the N-terminus. Eachcompound in FIGS. 9A-H is amidated at the C-terminal amino acid. FIG. 9Iis a graph showing the change in blood glucose in mice administered thecompounds shown in FIGS. 9B, 9C, 9D, 9F, 5A, and 1C based on the in vivoblood glucose assay described in Example 17. The compounds were injectedIP at t=0 immediately following a baseline sample in 2-hour fastedNIH/Swiss mice. Blood glucose samples were taken at t=30, 60, 120, 180,and 240 minutes with a ONETOUCH® ULTRA® (LifeScan, Inc., Milpitas,Calif.).

FIGS. 10A-I show the exendin analog in FIG. 5A having a modification atHis¹ at the N-terminus. R₄ is Xaa₂Xaa₃GTFTSDLSKQLEEEAVRLFIEFLKN-NH₂,where Xaa₂ is Gly, dAla, or Aib; and Xaa₃ is Glu or Pro. Alternatively,R₄ is Xaa₂Xaa₃GTFTSDLSKQLEEEA VRLFIEWLKNGGPSSGAPPPS-NH₂, where Xaa₂ isGly, dAla, or Aib; and Xaa₃ is Glu or Pro; provided that Xaa₃ is not Gluwhen Xaa₂ is Gly, or Xaa₂ is not Gly when Xaa₃ is Glu. Alternatively, R₄is Xaa₂Xaa₃GTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS-OH, where Xaa₂ is Gly,dAla, or Aib; and Xaa₃ is Glu or Pro.

FIGS. 11A-B show exendin-4 of FIG. 1A having a modification at His¹ atthe N-terminus. R₂ is GEGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂.

FIG. 12 shows the compound of FIG. 10E with an amino acid substitutionof Trp²⁵. The compound in FIG. 12 is amidated at the C-terminal aminoacid residue.

FIG. 13 shows an exendin analog having a modification at His¹Gly²Glu³.The compound in FIG. 13 is amidated at the C-terminal amino acidresidue.

FIG. 14: FIG. 14A is a generic structure (where Xaa₂ is, e.g., Gly,dAla, or Aib; and the other substituents are defined herein) of anexendin analog of FIG. 1A, 2A, or 5A having modifications atHis¹Gly²Glu³ at the N-terminus, where R is a peptide, such as any one ofthe following: GTFTSDLSKQLEEEAVRLFIEFLKN-NH₂;GTFTSDLSKQLEEEAV-RLFIEWLKQGGPSKEIIS-OH; orGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂. Aib is α-methylalanine. FIGS.14B-C are examples of compounds from the structure in FIG. 14A that havemodifications at His¹ and Glu³. The compounds in FIGS. 14B-C areamidated at the C-terminal amino acid residue. FIG. 14D provides thegeneric structure (where Xaa₂ is Gly, dAla, or Aib; and the othersubstituents are defined herein) of an N-terminus conformationallyconstrained compound. FIGS. 14E-R are exendin analogs comprising anN-terminus conformationally constrained compound. R₁ isGTFTSDLSKQLEEEAVRLF IEWLKQGGPSKEIIS-OH. R₄ isGTFTSDLSKQLEEEAVRLFIEFLKN-NH₂. R₅ isPGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂.

FIGS. 15A-D are exendin analogs that comprise isomers of nipecotic acidas the N-terminus conformationally constrained compound. R₁ isFTSDLSKQLEEEAVRLFI EWLKQGGPSKEIIS-NH₂.

FIG. 16: FIGS. 16A-H are exendin analogs containing a modification atthe N-terminus. R₁ is FTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS-OH. R₂ isFTSDLSKQLE EEAVRLFIEWLKNGGPSSGAPPPS-NH₂. R₃ isFTSDVSSYLEGQAAKEFIAWLVKGRG-NH₂. FIGS. 16I-L are exendin analogscontaining a modification at the N-terminus. The modification isdesigned to mimic amino acid residues His¹Gly²Glu³. R₄ is GTFTSDLSKQLEEEAVRLFIEFLKN-NH₂.

FIG. 17: FIGS. 17A-F are exendin analogs containing athiazolidine-proline peptide mimetics at Gly²Glu³. R₄ isGTFTSDLSKQLEEEAVRLFIEFLKN-NH₂. FIGS. 17G-N are exendin analogs having amodification at His¹ and containing a thiazolidine peptide mimetic atGly²Glu³. R₁, R₂, and R₃ are each independently hydrogen, methyl, orethyl. In this embodiment, Xaa₃ is Glu, Asp, Pro, or Gly. R₄ isGTFTSDLSKQLEEEAVRLFIEFLKN-NH₂.

FIG. 18: FIG. 18A is a process for preparing (5,5)-Glu-Gly-OH, adipeptide mimetic that can be used to induce a β-turn conformationalconstraint, for example, at the N-terminus in a GLP-1 receptor agonistcompound. FIG. 18B is a generic structure of the compound that can beproduced by the process shown in FIG. 18A. The skilled artisan canchoose compounds with different stereochemistries during the reactionprocess to provide for various stereochemistries in the final product. *represents a chiral carbon atom.

FIG. 19: FIG. 19A is a process for preparing γ-lactam Glu-Gly-OH, adipeptide mimetic that can be used to induce a β-turn conformationalconstraint, for example, at the N-terminus in a GLP-1 receptor agonistcompound. FIG. 19B is a generic structure of the compound that can beproduced by the process shown in FIG. 19A. The skilled artisan canchoose compounds with different stereochemistries during the reactionprocess to provide for various stereochemistries in the final product. *represents a chiral carbon atom.

FIG. 20: FIG. 20A is a process for preparing (6,5)-Asp-Gly-OH, adipeptide mimetic that can be used to induce β-turn conformationalconstraint, for example, at the N-terminus in a GLP-1 receptor agonistcompound. FIG. 20B is a generic structure of the compound that can beproduced by the process shown in FIG. 20A. The skilled artisan canchoose compounds with different stereochemistries during the reactionprocess to provide for various stereochemistries in the final product. *represents a chiral carbon atom.

FIG. 21: FIG. 21A is a process for preparing δ-lactam Asp-Gly-OH andAsp-Ala-OH, both of which are dipeptide mimetics that can be used toinduce β-turn conformational constraint, for example, at the N-terminusin a GLP-1 receptor agonist compound. FIG. 21B is a generic structure ofthe compound that can be produced by the process shown in FIG. 21A. Theskilled artisan can choose compounds with different stereochemistriesduring the reaction process to provide for various stereochemistries inthe final product. * represents a chiral carbon atom.

FIG. 22 is a process for preparing a peptide mimetic which can be usedto induce a β-turn conformational constraint, for example, at theN-terminus in a GLP-1 receptor agonist compound.

DETAILED DESCRIPTION

“GLP-1 receptor agonist compounds” refer to compounds that elicit abiological activity of an exendin reference peptide (e.g., exendin-4) ora GLP-1(7-37) reference peptide when evaluated by art-known measuressuch as receptor binding studies or in vivo blood glucose assays asdescribed, e.g., Examples 16 and 17, and by Hargrove et al, RegulatoryPeptides, 141:113-119 (2007), the disclosure of which is incorporated byreference herein. GLP-1 receptor agonist compounds include, for example,native exendins, exendin analogs, native GLP-1, GLP-1 analogs,GLP-1(7-37), and GLP-1(7-37) analogs.

The term “exendin” includes naturally occurring (or synthetic versionsof naturally occurring) exendin peptides that are found in the salivarysecretions of the Gila monster. Exendin-3(HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂) is present in the salivarysecretions of Heloderma horridum and exendin-4 (FIG. 1A) is present inthe salivary secretions of Heloderma suspectum. Exendins include theamidated forms, the acid form, the pharmaceutically acceptable saltform, and any other physiologically active form of the molecule. In oneembodiment, the term exendin can be used interchangeably with the term“exendin agonist.”

“Exendin analog” refers to peptides, peptides containing peptidemimetics, amino acid substitutions, and/or other modifications, peptidescontaining the N-terminus conformationally constrained compoundsdescribed herein, and/or other chemical moieties, or other compoundswhich elicit a biological activity similar to that of an exendinreference peptide (e.g., exendin-4), when evaluated by art-knownmeasures such as receptor binding assays or in vivo blood glucose assaysas described, e.g., Examples 16 and 17, and by Hargrove et al,Regulatory Peptides, 141:113-119 (2007), the disclosure of which isincorporated by reference herein. Preferably, the exendin analogs willbind in such receptor binding assays with an affinity of less than 1 μM;an affinity of less than 5 nM; an affinity of less than 1 nM, or anaffinity of less than 0.1 nM. In one embodiment, the term “exendinanalog” refers to a peptide that has an amino acid sequence with 1, 2,3, 4, 5, 6, 7, or 8 amino acid substitutions, insertions, deletions, ora combination of two or more thereof, when compared to the amino acidsequence of exendin-4 shown in FIG. 1A. In other embodiment, the term“exendin analog” refers to a peptide that has at least 85%, at least88%, at least 90%, at least 93%, at least 95%, or at least 98% sequenceidentity to the amino acid sequence of exendin-4 shown in FIG. 1A.Exendin analogs include the amidated forms, the acid form, thepharmaceutically acceptable salt form, and any other physiologicallyactive form of the molecule. In one embodiment, the term exendin analogcan be used interchangeably with the term “exendin agonist analog.”

“GLP-1(7-37) analogs” refers to peptides, peptides containing peptidemimetics and/or other modifications, peptides containing the N-terminusconformationally constrained compounds described herein, and/or otherchemical moieties, or other compounds which elicit a biological activitysimilar to that of GLP-1(7-37), when evaluated by art-known measuressuch as receptor binding assays or in vivo blood glucose assays asdescribed, e.g., Examples 16 and 17, and by Hargrove et al, RegulatoryPeptides, 141:113-119 (2007), the disclosure of which is incorporated byreference herein. In one embodiment, the term “GLP-1(7-37) analog”refers to a peptide that has an amino acid sequence with 1, 2, 3, 4, 5,6, 7, or 8 amino acid substitutions, insertions, deletions, or acombination of two or more thereof, when compared to the amino acidsequence of GLP-1(7-37). In one embodiment, the GLP-1(7-37) analog isGLP-1(7-36). GLP-1(7-37) analogs include the amidated forms, the acidform, the pharmaceutically acceptable salt form, and any otherphysiologically active form of the molecule.

“N-Terminus conformationally constrained GLP-1 receptor agonistcompounds” refers to compounds in which one, two, three, or four of theamino acid residues at positions 1-4 at the N-terminus of “GLP-1receptor agonist compounds” (e.g., exendins, exendin analogs, GLP-1,GLP-1 analogs, GLP-1(7-37), GLP-1(7-37) analogs) have been modified orsubstituted with amino acids (e.g., natural and/or non-natural aminoacids), peptidomimetics, or beta-turn dipetidemimetics. Thissubstitution(s) or modification(s) to the N-terminus of the parent GLP-1receptor agonist compound changes the flexible random coil structure ofthis specific region into a more rigid secondary structure withbeta-turn characteristics.

The glycine residues at positions 2 and 4 at the N-terminus of exendin-4may indicate the presence of a β-turn in this region. In order toproduce a conformationally constrained N-terminus, exendin analogshaving a mimetic or other structural modification that restricted theconformational flexibility of the His¹ side chain were synthesized.Restricting the flexibility of the His¹ side chain was hypothesized toprovide structural information about the possible bioactive conformationof GLP-1 receptor agonist compounds and thus enhance GLP-1 receptorbinding, in vivo blood glucose lowering activity, and enzymaticstability.

An Ala scan of exendin-4 showed that the Glu³ residue was important forbiological activity. Additionally, Glu³ or Asp³ are present in manymembers of the super-family of glucagon-related peptides, whichindicates the importance of an acidic side chain at that residue. It waspostulated that the negative charge of Glu³ or Asp³ interacted throughan ionic bond with the positive charge of the His¹ side chain toposition the key imidazole ring of the His¹ side chain in the rightspace for interaction with the GLP-1 receptor. It was thus proposed thata β-turn would be formed by the sequence Gly²Glu³Gly⁴Thr⁵ in thesuper-family of glucagon related peptides, such as exendin-4 and exendinanalogs.

In order to maintain the negative charge of Glu³, thought to beessential for biological activity, β-turn peptide mimetics weresynthesized to mimic the amino acid residues Glu³Gly⁴. In oneembodiment, the disclosure provides N-terminus conformationallyconstrained compounds of Formula (F):

Xaa₁Xaa₂Xaa₃-Z;

wherein:Xaa₁ is a compound of Formula (1), as described herein;Xaa₂ is Gly, Ala, dAla, or Aib;

Xaa₃ is:

-   -   wherein * indicates a chiral carbon atom; and R₂ is hydrogen or        a C₁₋₄ alkyl (e.g., methyl, ethyl); and

Z is: (i) OH;

(ii) NH₂;

(iii) TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKN-Z₁;

(iv) TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKNGGPSSGAPPPS-Z₁;

(v) TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LK-R₁₀-Z₁; or

(vi) TFTSDVSSYXaa₁₄EGQAAKEFIAXaa₂₅LVXaa₂₈GRXaa₃₁-Z₁;

-   -   wherein:    -   Z₁ is OH or NH₂;    -   Xaa₁₄ is Leu or Met;    -   Xaa₂₅ is Phe or Trp;    -   Xaa₂₈ is Lys or Arg;    -   Xaa₃₁ is Gly or absent; and    -   R₁₀ is QGGPSKEIIS; QGGPSSGAPPPS; NG; NGG; NGGP; NGGPS; NGGPSS;        NGGPSSG; NGGPSSGA; NGGPSSGAP; NGGPSSGAPP; NGGPSSGAPPP;        NGGPSSGAPPS; NGGPSSGAPPSK; NGGPSSGAPPS(K)₂₋₆; NGGPSSGAPPPSK; or        NK.

When Z is OH or NH₂, the compounds of Formula (F) are N-terminusconformationally constrained compounds. When Z is (iii), (iv), (v) or(vi), the compounds of Formula (F) are N-terminus conformationallyconstrained GLP-1 receptor agonist compounds.

Exemplary compounds of Formula (F) include the compounds in FIGS. 15A-Dand 16A-H, each of which may be optionally amidated at the C-terminalamino acid residue. The reaction schemes for preparing the compounds areshown, e.g., in FIGS. 18-20.

Additional studies were undertaken to restrict the N-terminusconformation of GLP-1 receptor agonist compounds and it was unexpectedlydiscovered that the β-turn in GLP-1 receptor agonist compounds, such asexendin and exendin analogs, was provided by His¹Gly²Glu³Gly⁴. Thus,GLP-1 receptor agonist compounds were produced to constrain or mimic aβ-turn defined by residues His¹Gly²Glu³Gly⁴ in exendin-4 and other GLP-1receptor agonist compounds; or to constrain or mimic a β-turn defined byresidues His¹Ala²Glu³Gly⁴ in GLP-1, GLP-1 analogs, GLP-1(7-37), orGLP-1(7-37) analogs.

Provided herein are N-terminus conformationally constrained compounds ofFormula (A): Xaa₁Xaa₂Xaa₃Xaa₄-Z.

In one embodiment, the disclosure provides the compound of Formula (A):

Xaa₁Xaa₂Xaa₃Xaa₄-Z;

wherein:

-   Xaa₁ is a compound of Formula (1):

-   -   wherein R₂₀ and R₂₁ are each independently a single bond or a        carbon atom; R₂₃, R₂₄, R₂₅ and R₂₆ are each independently        absent, hydrogen, hydroxyl, C₁₋₄ alkyl, carboxyl, or C₁₋₄        alkoxy;        is a single bond or a double bond; and R₂₁ is a chiral or        achiral carbon atom;

-   Xaa₂ is Gly, dAla, Aib, Ala, Val, NMeAla, a compound of Formula (3),    as described herein; or a compound of Formula (4) and described    herein; and Xaa₂ is absent when Xaa₃ is:

-   Xaa₃ is Pro; a compound of Formula (2), as described herein; a    compound of Formula (3), as described herein; a Compound of Formula    (4), as described herein;

-   Xaa₄ is Gly, dAla, or Aib; and-   Z is OH or NH₂. In other embodiments, Xaa₃ is Pro. In other    embodiments, Xaa₂ is dAla, Aib, Ala, Val, NMeAla, a compound of    Formula (3), or a compound of Formula (4).

In other embodiments, the disclosure provides the compound of Formula(A):

Xaa₁Xaa₂Xaa₃Xaa₄-Z;

wherein:

-   Xaa₁ is

-   Xaa₂ is Gly, dAla, Aib, Ala, Val, NMeAla, a compound of Formula (3),    as described herein; or a compound of Formula (4), as described    herein; and Xaa₂ is absent when Xaa₃ is:

-   Xaa₃ is Glu; Pro; a compound of Formula (2), as described herein; a    compound of Formula (3), as described herein; a Compound of Formula    (4), as described herein;

-   Xaa₄ is Gly, dAla, or Aib; and-   Z is OH or NH₂.

Also provided herein are N-terminus conformationally constrained GLP-1receptor agonist compounds of Formula (B)-(E):

(B) Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKN-Z; (C)Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LK-R₁₀-Z; (D)Xaa₁Xaa₂aXaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKNGGPSSGAPPPS-Z; (E)Xaa₁Xaa₂Xaa₃Xaa₄TFTSDVSSYXaa₁₄EGQAAKEFIAXaa₂₅LVXaa₂₈GRXaa₃₁-Z.The substituents for the compounds of Formula (A), (B), (C), (D), and(E) are as follows:

-   Xaa₁ is a compound of Formula (1), as described herein;-   Xaa₂ is Gly; dAla; Aib; Ala; Val; NMeAla; a compound of Formula (3),    as described herein; or a compound of Formula (4), as described    herein; and Xaa₂ is absent when Xaa₃ is:

-   Xaa₃ is Pro; Glu; Asp; a compound of Formula (2), as described    herein; a compound of Formula (3), as described herein; a Compound    of Formula (4), as described herein;

-   Xaa₄ is Gly, dAla, or Aib;-   Xaa₁₄ is Leu or Met;-   Xaa₂₅ is Phe or Trp;-   Xaa₂₈ is Lys or Arg;-   Xaa₃₁ is Gly or absent;-   R₁₀ is QGGPSKEIIS; QGGPSSGAPPPS; NG; NGG; NGGP; NGGPS; NGGPSS;    NGGPSSG; NGGPSSGA; NGGPSSGAP; NGGPSSGAPP; NGGPSSGAPPP; NGGPSSGAPPS;    NGGPSSGAPPSK; NGGPSSGAPPS(K)₂₋₅; NGGPSSGAPPPSK; or NK; and-   Z is OH or NH₂.

The compounds of Formula (A)-(E) may optionally be in the form of apharmaceutically acceptable salt.

The compound of Formula (1) is:

wherein R₂₀ and R₂₁ are each independently a single bond or a carbonatom; R₂₃, R₂₄, R₂₅ and R₂₆ are each independently absent, hydrogen,hydroxyl, C₁₋₄ alkyl, carboxyl, amino, or C₁₋₄ alkoxy;

is a single bond or a double bond; and R₂₁ is a chiral or achiral carbonatom.

In one embodiment for the compound of Formula (1), R₂₀ and R₂₁ are eachindependently a single bond or a carbon atom; R₂₃ and R₂₄ are eachindependently absent, hydrogen, hydroxy, a C₁₋₄ alkyl, carboxyl, or aC₁₋₄ alkoxy; R₂₅ and R₂₆ are each independently absent, hydrogen,hydroxy, a C₁₋₄ alkyl, carboxyl, amino, or a C₁₋₄ alkoxy;

is a single bond or a double bond; and R₂, is a chiral or achiral carbonatom.

In one embodiment for the compound of Formula (1), R₂₀ and R₂₁ are eachindependently a single bond or a carbon atom; R₂₃ and R₂₄ are eachindependently absent, hydrogen, hydroxy, methyl, ethyl, or carboxyl; R₂₅and R₂₆ are each independently absent or hydrogen;

is a single bond or a double bond; and R₂₁ is a chiral or achiral carbonatom.

In one embodiment, the compound of Formula (1) is a compound of Formula(1a), (1b), (1c), (1d), (1e), (1f, (1g), (1h), (1j), (1k), (1m), or(1n):

In one embodiment, the compound of Formula (1) is a compound of Formula(1a), (1b), (1c), (1d), (1e), (1f, (1g), (1h), (1j), (1k), or (1m).

In one embodiment, the compound of Formula (1) is a compound of Formula(1a), (1b), (1c), (1d), (1e), (1f, (1g), (1h), or (1k).

In one embodiment, the compound of Formula (1) is a compound of Formula(1j) or (1m).

In one embodiment, the compound of Formula (1) is a compound of Formula(1n).

The compounds of Formula (2) and Formula (3) are:

wherein Y₁ and Z₁ are each independently a single bond, a carbon, or asulfur; and W₁, W₂ and W₃ are each independently selected from hydrogen,C₁₋₆ alkyl, C₁₋₆ alkoxy, hydroxyl, and amino; and when one Y₁ or Z₁ issulfur, the sulfur may be bonded to two oxygen atoms to form a sulfonylgroup; and

is

or

.

In one embodiment for the Compounds of Formula (2) and (3), Y₁ and Z₁are each independently a single bond, a carbon, or a sulfur; and W₁, W₂and W₃ are each independently selected from hydrogen, C₁₋₄ alkyl, C₁₋₄alkoxy, and amino; and when one Y₁ or Z₁ is sulfur, the sulfur may bebonded to two oxygen atoms to form a sulfonyl group; and

is

or

.

In one embodiment for the Compounds of Formula (2) and (3), Y₁ and Z₁are each independently a single bond, a carbon, or a sulfur; and W₁, W₂and W₃ are each independently selected from hydrogen, C₁₋₄ alkyl, andC₁₋₄ alkoxy; and when one Y₁ or Z₁ is sulfur, the sulfur may be bondedto two oxygen atoms to form a sulfonyl group; and

is

or

.

In one embodiment for the Compounds of Formula (2) and (3),

is

.

In one embodiment for the Compounds of Formula (2), Y₁ and Z₁ are eachindependently a single bond, a carbon, or a sulfur; and W₁, W₂ and W₃are each independently selected from the group consisting of hydrogen,methyl, ethyl, and propyl; and

is

.

In one embodiment for the Compounds of Formula (3), Y₁ and Z₁ are eachindependently a single bond or carbon; and W₁, W₂ and W₃ are eachindependently selected from the group consisting of hydrogen, methyl,ethyl, and propyl; and

is

.

In one embodiment, the compound of Formula (2) is a compound of Formula(2Z):

wherein R₄₀, R₄₁, and R₄₂ are each independently selected from the groupconsisting of hydrogen and C₁₋₄ alkyl (preferably methyl or ethyl).Exemplary compounds of Formula (2Z) include compounds of Formula (2d),(2e), (2f, (2g), and (2h) described below.

In one embodiment, the compound of Formula (2) is a compound of Formula(2a), (2b), (2c), (2d), (2e), (2f, (2g), (2h), or (2j):

In one embodiment, the compound of Formula (2) is a compound of Formula(2d), (2e), (2f, (2g), or (2h).

In one embodiment, the compound of Formula (3) is a compound of Formula(3a), (3b), or (3c):

The compound of Formula (4) is:

wherein R₃₀, R₃₁, and R₃₂ are each independently hydrogen or a C₁₋₄alkyl; or R₃₀ and R₃₁, together with the nitrogen¹ and the carbon², forma 5-membered or 6-membered heterocyclic ring; or R₃₁ and R₃₂, togetherwith the carbon², form a 3-, 4-, or 5-membered carbocyclic ring.

In one embodiment for the compound of Formula (4), R₃₀, R₃₁, and R₃₂ areeach independently hydrogen, methyl, or ethyl; or R₃₀ and R₃₁, togetherwith the nitrogen¹ and carbon², form a 5-membered or 6-memberedheterocyclic ring; or R₃₁ and R₃₂, together with the carbon², form a 3-,or 4-membered carbocyclic ring;

In one embodiment, the compound of Formula (4) is a compound of Formula(4a), (4b), (4c), (4d), or (4e):

In one embodiment for the Compounds of Formula (A)-(F), Xaa₂ is dAla,Aib, Ala, Val, or NMeAla.

In one embodiment for the Compounds of Formula (A)-(F), Xaa₂ is acompound of Formula (3).

In one embodiment for the Compounds of Formula (A)-(F), Xaa₂ is acompound of Formula (4).

In one embodiment for the Compounds of Formula (A)-(F), Xaa₂ is Gly.

In one embodiment for the Compounds of Formula (A)-(F), Xaa₂ is dAla.

In one embodiment for the Compounds of Formula (A)-(F), Xaa₂ is dAla orAib.

In one embodiment for the Compounds of Formula (A)-(F), Xaa₃ is Pro.

In one embodiment for the Compounds of Formula (A)-(F), Xaa₃ is Glu.

In one embodiment for the Compounds of Formula (A)-(F), Xaa₃ is acompound of Formula (2), as described herein.

In one embodiment for the Compounds of Formula (A)-(F), Xaa₃ is acompound of Formula (3), as described herein.

In one embodiment for the Compounds of Formula (A)-(F), Xaa₂ is absentand Xaa₃ is

In one embodiment for the Compounds of Formula (A)-(F), Xaa₄ is Gly ordAla.

In one embodiment for the Compounds of Formula (A)-(F), Z is NH₂.

For the Compounds of Formula (A)-(F): in one embodiment R₁₀ isQGGPSKEIIS; in one embodiment R₁₀ is NG; in one embodiment R₁₀ is NGG;in one embodiment R₁₀ is NGGP; in one embodiment R₁₀ is NGGPS; in oneembodiment R₁₀ is NGGPSS; in one embodiment R₁₀ is NGGPSSG; in oneembodiment R₁₀ is NGGPSSGA; in one embodiment R₁₀ is NGGPSSGAP; in oneembodiment R₁₀ is NGGPSSGAPP; in one embodiment R₁₀ is NGGPSSGAPPP; inone embodiment R₁₀ is NGGPSSGAPPS; in one embodiment R₁₀ isNGGPSSGAPPSK; in one embodiment R₁₀ is NGGPSSGAPPS(K)₂₋₅; in oneembodiment R₁₀ is NGGPSSGAPPPSK; and in one embodiment R₁₀ is NK; and inone embodiment R₁₀ is QGGPSSGAPPPS.

For the Compounds of Formula (A)-(F): in one embodiment Xaa₁₄ is Met andXaa₂₅ is Trp; in one embodiment Xaa₁₄ is Leu and Xaa₂₅ is Phe; in oneembodiment Xaa₁₄ is Met and Xaa₂₅ is Phe; and in one embodiment Xaa₁₄ isLeu and Xaa₂₅ is Trp.

With respect to the compounds of Formula (D), Xaa₂, Xaa₃, Xaa₁₄, andXaa₂₅ cannot simultaneously be Gly, Glu, Met, and Trp, respectively,except when the compound of Formula (1) is a compound of Formula 1(j) or1(m). Thus, when Xaa₂, Xaa₃, Xaa₁₄, and Xaa₂₅ are Gly, Glu, Met, andTrp, respectively, the compound of Formula (D) may be one of thefollowing:

In one embodiment, the N-terminus conformationally constrained GLP-1receptor agonist compound may be Pro³-exendin-4; Pro³,Leu¹⁴-exendin-4;Pro³,Leu¹⁴,Phe²⁵-exendin-4; Pro³-exendin-4(1-28);Pro³,Leu¹⁴-exendin-4(1-28); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-28);Pro³-exendin-4(1-36); Pro³,Leu¹⁴-exendin-4(1-36);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-36); orHGPGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS, each of which may optionally beamidated and which may optionally be in the form of a pharmaceuticallyacceptable salt.

In one embodiment, the N-terminus conformationally constrained GLP-1receptor agonist compound may be Pro³-exendin-3; Pro³-exendin-4(1-29);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-29); Pro³-exendin-4(1-30);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-30); Pro³-exendin-4(1-31);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-31); Pro³-exendin-4(1-32);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-32); Pro³-exendin-4(1-33);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-33); Pro³-exendin-4(1-34);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-34); Pro³-exendin-4(1-35); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-35); Pro³-exendin-4(1-37);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-37); Pro³-exendin-4(1-38); orPro³,Leu¹⁴,Phe²⁵-exendin-4(1-38), each of which may optionally beamidated and which may optionally be in the form of a pharmaceuticallyacceptable salt.

Examples of the compounds of Formula (A), (B), (C), (D), and (E) areshown in FIGS. 1B-C, 2A-U, 3A-G, 5B-G, 6A-E, 8A-H, 9A-H, 10A-I, 11A-B,12, 13, 14A-Q, 15C-D, 16I-L, and 17A-N.

The N-terminus conformationally constrained compounds (e.g., compoundsof Formula (A) and (F)) and the N-Terminus conformationally constrainedGLP-1 receptor agonist compounds (e.g., compounds of Formula (B)-(F))described herein (collectively referred to as “the compounds”) canoptionally be covalently linked to one or more polymers to providebeneficial biological properties to the compounds. Such beneficialproperties may include conferring another therapeutic property to thecompounds; increasing the in vivo half life of the compounds; decreasingthe rate of clearance of the compounds by the kidney; decreasing theimmunogenicity of the compounds; decreasing the proteolysis rate of thecompounds; or increasing the stability of the compounds. Exemplarypolymers that can be covalently linked to the compounds includepeptides, polyethylene glycols, albumin, fatty acids, dextran, polyaminoacids, alkyl chains, immunoglobulins, signaling moieties, gelatin,polyvinyl pyrrolidone, polyvinyl alcohol,N-(2-hydroxypropyl)-methacrylamide, and the like. In one embodiment, twoor more polymers (e.g., peptides, polyethylene glycols, albumin, fattyacids, dextran, polyamino acids, alkyl chains, immunoglobulins, gelatin,polyvinyl pyrrolidone, polyvinyl alcohol,N-(2-hydroxypropyl)-methacrylamide) are covalently attached together andlinked to the N-terminus conformation constrained compounds describedherein. For example, the two more polymers that are linked together maybe polyethylene glycol(s) and fatty acid(s) or a peptide, polyethyleneglycol(s), and fatty acids.

In one embodiment, the compounds are linked to another peptide toprovide additional therapeutic benefits. Such peptides include amylin,amylin agonist analogs (e.g., amylin analogs that function as amylinagonists), PYY, PYY analogs, GIP, GIP analogs, leptin, metraleptin,leptin analogs, metraleptin analogs, and the like. Hybrid peptidescomprising the compounds and another therapeutic peptide are described,for example, in WO 2005/077072 and WO 2007/022123, the disclosures ofwhich are incorporated by reference herein.

In one embodiment, compounds are linked to one, two, or threepolyethylene glycols. In one embodiment, the compounds are linked to onepolyethylene glycol. The polyethylene glycol can have a molecular weightfrom about 200 daltons to about 80,000 daltons; from about 5,000 fromabout 10,000 daltons to about 60,000 daltons; from about 10,000 daltonsto about 50,000 daltons; or from about 15,000 daltons to about 40,000daltons. The polyethylene glycol may be linear or branched.

In one embodiment, compounds are linked to one or two polyethyleneglycols, where the polyethylene glycol is further linked to a lipophilicmoiety. In one embodiment, the polyethylene glycol may have a molecularweight from about 200 to about 7,000 daltons or from about 500 to about5,000 daltons. The lipophilic moiety may be an alkyl group (e.g., C₁₋₂₀alkyl group; C₁₋₁₀ alkyl group; C₁₋₆ alkyl group; C₁₋₄ alkyl group), afatty acid (e.g., C₄₋₂₈ fatty acid; C₄₋₂₀ fatty acid; C₄₋₁₀ fatty acid),cholesteryl, adamantyl, and the like. The alkyl group may be linear orbranched, preferably linear. In one embodiment, the fatty acid is anacetylated fatty acid or an esterified fatty acid. The -(polyethyleneglycol)-(lipophilic moiety) may be linked to the compound at aC-terminal amino acid residue, an N-terminal amino acid residue, aninternal amino acid residue (e.g., an internal Lys amino acid residue),or a combination thereof (e.g., the compound is linked at the N-terminaland C-terminal amino acid residues via a lysine residue). Examplarypeptides linked to such groups are shown in Example 20.

In one embodiment, the compounds are linked to a polyamino acid.Exemplary polyamino acids include poly-lysine, poly-aspartic acid,poly-serine, poly-glutamic acid, and the like. The polyamino acid may bein the D or L form, preferably the L form. The polyamino acids maycomprise from 1 to 12 amino acid residues; from 2 to 10 amino acidresidues; or from 2 to 6 amino acid residues.

In one embodiment, compounds are linked to a fatty acid. The fatty acidmay be a C₄-C₂₈ fatty acid chain, a C₈-C₂₄ fatty acid chain, or aC₁₀-C₂₀ fatty acid chain. In one embodiment, the fatty acid is anacetylated fatty acid. In one embodiment, the fatty acid is anesterified fatty acid.

In one embodiment, the compounds are linked to albumin. The albumin maybe a recombinant albumin, serum albumin, or recombinant serum albumin.In another embodiment, the compounds are linked to an albumin-fatty acid(i.e., an albumin linked to a fatty acid).

In one embodiment, the compounds are linked to an immunoglobulin or animmunoglobulin Fc region. The immunoglobulin may be IgG, IgE, IgA, IgD,or IgM. In one embodiment, the compounds are linked to an IgG Fc regionor an IgM Fc region. The immunoglobulin Fc region is (i) the heavy chainconstant region 2(C_(H)2) of an immunoglobulin; (ii) the heavy chainconstant region 3(C_(H)3) of an immunoglobulin; or (iii) both the heavychain constant regions 2(C_(H)2) and 3(C_(H)3) of an immunoglobulin. Theimmunoglobulin Fc region may further comprise the hinge region at theheavy chain constant region. Other embodiments for the immunoglobulin Fcregion that can be linked to exendin analog peptides are described in WO2008/082274, the disclosure of which is incorporated by referenceherein.

In one embodiment, the compounds are linked to one or more signallingmoieties. Exemplary signalling moieties include, biotin, antigens,antibodies, receptors, enzymes, chemiluminescent groups, photoreactivegroups, fluorescent groups, heavy metal-containing compounds (e.g.,ferritin), and the like.

When the compounds described herein are covalently linked to one or morepolymers, such as those described herein, any linking group known in theart can be used. The linking group may comprise any chemical group(s)suitable for linking the peptide to the polymer. Alternatively,compounds can be directly attached to the polymer without any linkinggroup. Exemplary linking groups include amino acids, maleimido groups,dicarboxylic acid groups, succinimide groups, or a combination of two ormore thereof. Methods for linking peptides to one or more polymers areknown in the art and described, for example, in U.S. Pat. No. 6,329,336;U.S. Pat. No. 6,423,685; U.S. Pat. No. 6,924,264; WO 2005/077072, WO2007/022123, WO 2007/053946; WO 2008/058461; and WO 2008/082274, thedisclosures of which are incorporated by reference herein.

The compounds described herein may be prepared using biological,chemical, and/or recombinant DNA techniques that are known in the art.Exemplary methods are described in U.S. Pat. No. 6,872,700; WO2007/139941; WO 2007/140284; WO 2008/082274; WO 2009/011544; and USPublication No. 2007/0238669, the disclosures of which are incorporatedherein by reference. Other methods for preparing the compounds are setforth herein.

The compounds described herein may be prepared using standardsolid-phase peptide synthesis techniques, such as an automated orsemiautomated peptide synthesizer. Typically, using such techniques, analpha-N-carbamoyl protected amino acid and an amino acid attached to thegrowing peptide chain on a resin are coupled at room temperature in aninert solvent (e.g., dimethylformamide, N-methylpyrrolidinone, methylenechloride, and the like) in the presence of coupling agents (e.g.,dicyclohexylcarbodiimide, 1-hydroxybenzo-triazole, and the like) in thepresence of a base (e.g., diisopropylethylamine, and the like). Thealpha-N-carbamoyl protecting group is removed from the resultingpeptide-resin using a reagent (e.g., trifluoroacetic acid, piperidine,and the like) and the coupling reaction repeated with the next desiredN-protected amino acid to be added to the peptide chain. SuitableN-protecting groups are well known in the art, such ast-butyloxycarbonyl (tBoc) fluorenylmethoxycarbonyl (Fmoc), and the like.The solvents, amino acid derivatives and 4-methylbenzhydryl-amine resinused in the peptide synthesizer may be purchased from Applied BiosystemsInc. (Foster City, Calif.).

Solid phase peptide synthesis may be carried out with an automaticpeptide synthesizer (Model 430A, Applied Biosystems Inc., Foster City,Calif.) using the NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry(See Applied Biosystems User's Manual for the ABI 430A PeptideSynthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70, AppliedBiosystems, Inc., Foster City, Calif.) with capping. Boc-peptide-resinsmay be cleaved with HF (−5° C. to 0° C., 1 hour). The peptide may beextracted from the resin with alternating water and acetic acid, and thefiltrates lyophilized. The Fmoc-peptide resins may be cleaved accordingto standard methods (e.g., Introduction to Cleavage Techniques, AppliedBiosystems, Inc., 1990, pp. 6-12). Peptides may be also be assembledusing an Advanced Chem Tech Synthesizer (Model MPS 350, Louisville,Ky.).

Peptides may be purified by RP-HPLC (preparative and analytical) using aWaters Delta Prep 3000 system. A C4, C8 or C18 preparative column (10μ,2.2X25 cm; Vydac, Hesperia, Calif.) may be used to isolate peptides, andpurity may be determined using a C4, C8 or C18 analytical column (5μ,0.46X25 cm; Vydac). Solvents (A=0.1% TFA/water and B=0.1% TFA/CH₃CN) maybe delivered to the analytical column at a flow rate of 1.0 ml/min andto the preparative column at 15 ml/min. Amino acid analyses may beperformed on the Waters Pico Tag system and processed using the Maximaprogram. Peptides may be hydrolyzed by vapor-phase acid hydrolysis (115°C., 20-24 h). Hydrolysates may be derivatized and analyzed by standardmethods (Cohen et al, The Pico Tag Method: A Manual of AdvancedTechniques for Amino Acid Analysis, pp. 11-52, Millipore Corporation,Milford, Mass. (1989)). Fast atom bombardment analysis may be carriedout by M-Scan, Incorporated (West Chester, Pa.). Mass calibration may beperformed using cesium iodide or cesium iodide/glycerol. Plasmadesorption ionization analysis using time of flight detection may becarried out on an Applied Biosystems Bio-Ion 20 mass spectrometer.

The compounds described herein may also be prepared using recombinantDNA techniques using methods known in the art, such as Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor(1989). Non-peptide compounds may be prepared by art-known methods. Forexample, phosphate-containing amino acids and peptides containing suchamino acids, may be prepared using methods known in the art, such asdescribed in Bartlett et al, Biorg. Chem., 14:356-377 (1986).

The disclosure also provides pharmaceutical compositions comprising atleast one of the N-terminus conformationally constrained compounds orthe N-terminus conformationally constrained GLP-1 receptor agonistcompounds described herein and a pharmaceutically acceptable carrier.The N-terminus conformationally constrained compounds or the N-terminusconformationally constrained GLP-1 receptor agonist compounds can bepresent in the pharmaceutical composition in a therapeutically effectiveamount and can be present in an amount to provide a minimum blood plasmalevel for therapeutic efficacy.

Pharmaceutical compositions containing the compounds described hereinmay be provided for peripheral administration, such as parenteral (e.g.,subcutaneous, intravenous, intramuscular), topical, nasal, or oraladministration. Suitable pharmaceutically acceptable carriers and theirformulation are described in standard formulation treatises, such asRemington's Pharmaceutical Sciences by Martin; and Wang et al, Journalof Parenteral Science and Technology, Technical Report No. 10, Supp.42:2S (1988).

The compounds described herein can be provided in parenteralcompositions for injection or infusion. They can, for example, besuspended in water; an inert oil, such as a vegetable oil (e.g., sesame,peanut, olive oil, and the like); or other pharmaceutically acceptablecarrier. In one embodiment, the compounds are suspended in an aqueouscarrier, for example, in an isotonic buffer solution at a pH of about3.0 to 8.0, or about 3.0 to 5.0. The compositions may be sterilized byconventional sterilization techniques or may be sterile filtered. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH buffering agents. Useful buffers include for example, acetic acidbuffers. A form of repository or “depot” slow release preparation may beused so that therapeutically effective amounts of the preparation aredelivered into the bloodstream over many hours or days followingsubcutaneous injection, transdermal injection or other delivery method.The desired isotonicity may be accomplished using sodium chloride orother pharmaceutically acceptable agents such as dextrose, boric acid,sodium tartrate, propylene glycol, polyols (such as mannitol andsorbitol), or other inorganic or organic solutes. Sodium chloride ispreferred particularly for buffers containing sodium ions.

The compounds can also be formulated as pharmaceutically acceptablesalts (e.g., acid addition salts) and/or complexes thereof.Pharmaceutically acceptable salts are non-toxic salts at theconcentration at which they are administered. Pharmaceuticallyacceptable salts include acid addition salts such as those containingsulfate, hydrochloride, phosphate, sulfamate, acetate, citrate, lactate,tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, cyclohexylsulfamate and quinate. Pharmaceuticallyacceptable salts can be obtained from acids such as hydrochloric acid,sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid,lactic acid, tartaric acid, malonic acid, methanesulfonic acid,ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,cyclohexylsulfamic acid, and quinic acid. Such salts may be prepared by,for example, reacting the free acid or base forms of the product withone or more equivalents of the appropriate base or acid in a solvent ormedium in which the salt is insoluble, or in a solvent such as waterwhich is then removed in vacuo or by freeze-drying or by exchanging theions of an existing salt for another ion on a suitable ion exchangeresin.

Carriers or excipients can also be used to facilitate administration ofthe compounds. Examples of carriers and excipients include calciumcarbonate, calcium phosphate, various sugars such as lactose, glucose,or sucrose, or types of starch, cellulose derivatives, gelatin,vegetable oils, polyethylene glycols and physiologically compatiblesolvents.

If desired, solutions of the above compositions may be thickened with athickening agent such as methyl cellulose. They may be prepared inemulsified form, either water in oil or oil in water. Any of a widevariety of pharmaceutically acceptable emulsifying agents may beemployed including, for example, acacia powder, a non-ionic surfactant(such as a Tween), or an ionic surfactant (such as alkali polyetheralcohol sulfates or sulfonates, e.g., a Triton).

Compositions may be prepared by mixing the ingredients followinggenerally accepted procedures. For example, the selected components maybe simply mixed in a blender or other standard device to produce aconcentrated mixture which may then be adjusted to the finalconcentration and viscosity by the addition of water or thickening agentand possibly a buffer to control pH or an additional solute to controltonicity.

The therapeutically effective amount of the compounds described hereinto treat the diseases described herein will typically be from about 0.01μg to about 5 mg; about 0.1 μg to about 2.5 mg; about 1 mg to about 1mg; about 1 μg to about 50 μg; or about 1 μg to about 25 μg.Alternatively, the therapeutically effective amount of the GLP-1receptor agonist compounds may be from about 0.001 μg to about 100 μgbased on the weight of a 70 kg patient; or from about 0.01 μg to about50 μg based on the weight of a 70 kg patient. These therapeuticallyeffective doses may be administered once/day, twice/day, thrice/day,once/week, biweekly, or once/month, depending on the formulation. Theexact dose to be administered is determined, for example, by theformulation, such as an immediate release formulation or an extendedrelease formulation. For transdermal, nasal or oral dosage forms, thedosage may be increased from about 5-fold to about 10-fold.

The compounds described herein and pharmaceutical compositionscomprising the compounds are useful for treating diabetes. The diabetescan be Type I diabetes, Type II diabetes, or gestational diabetes. Themethods for treating diabetes provide administering to a patient in needthereof a therapeutically effective amount of one or more of theN-terminus conformationally constrained compounds or the N-terminusconformationally constrained GLP-1 receptor agonist compounds describedherein to treat diabetes in the patient.

The compounds described herein and pharmaceutical compositionscomprising the compounds are useful for treating insulin resistance andstimulating insulin release. The methods for treating insulin resistanceprovide administering to a patient in need thereof a therapeuticallyeffective amount of one or more of the N-terminus conformationallyconstrained compounds or the N-terminus conformationally constrainedGLP-1 receptor agonist compounds described herein to treat insulinresistance in the patient. The methods for treating stimulating insulinrelease provide administering to a patient in need thereof atherapeutically effective amount of one or more of the N-terminusconformationally constrained compounds or the N-terminusconformationally constrained GLP-1 receptor agonist compounds describedherein to stimulate insulin release in the patient.

The compounds described herein and pharmaceutical compositionscomprising the compounds are useful for treating postprandialhyperglycemia. The methods for treating postprandial hyperglycemiaprovide administering to a patient in need thereof a therapeuticallyeffective amount of one or more of the N-terminus conformationallyconstrained compounds or the N-terminus conformationally constrainedGLP-1 receptor agonist compounds described herein to treat postprandialhyperglycemia in the patient.

The compounds described herein and pharmaceutical compositionscomprising the compounds are useful for lowering blood glucose levelsand lowering HbA1c levels. The methods for lowering blood glucose levelsprovide administering to a patient in need thereof a therapeuticallyeffective amount of one or more of the N-terminus conformationallyconstrained compounds or the N-terminus conformationally constrainedGLP-1 receptor agonist compounds described herein to lower blood glucoselevels in the patient. In one embodiment, the blood glucose levels canbe fasting blood glucose levels. The methods for lowering HbA1c levelsprovide administering to a patient in need thereof a therapeuticallyeffective amount of one or more of the N-terminus conformationallyconstrained compounds or the N-terminus conformationally constrainedGLP-1 receptor agonist compounds described herein to lower HbA1c levelsin the patient. HbA1c levels are generally a long-term measure of apatient's blood glucose levels.

The compounds described herein and pharmaceutical compositionscomprising the compounds are useful for reducing gastric motility anddelaying gastric emptying. The methods for reducing gastric motilityprovide administering to a patient in need thereof a therapeuticallyeffective amount of one or more of the N-terminus conformationallyconstrained compounds or the N-terminus conformationally constrainedGLP-1 receptor agonist compounds described herein to reduce gastricmotility in the patient. The methods for delaying gastric emptyingprovide administering to a patient in need thereof a therapeuticallyeffective amount of one or more of the N-terminus conformationallyconstrained compounds or the N-terminus conformationally constrainedGLP-1 receptor agonist compounds described herein to delay gastricemptying in the patient.

The compounds described herein and pharmaceutical compositionscomprising the compounds are useful for reducing food intake, reducingappetite, increasing satiety, and reducing weight. The methods forreducing food intake provide administering to a patient in need thereofa therapeutically effective amount of one or more of the N-terminusconformationally constrained compounds or the N-terminusconformationally constrained GLP-1 receptor agonist compounds describedherein to reduce food intake in the patient. The methods for reducingappetite provide or increasing satiety administering to a patient inneed thereof a therapeutically effective amount of one or more of theN-terminus conformationally constrained compounds or the N-terminusconformationally constrained GLP-1 receptor agonist compounds describedherein to reduce appetite in the patient. The methods for treatingreducing weight provide administering to a patient in need thereof atherapeutically effective amount of one or more of the N-terminusconformationally constrained compounds or the N-terminusconformationally constrained GLP-1 receptor agonist compounds describedherein to reduce weight in the patient. In the methods described herein,the patient may be in need of a reduced intake in food, of a reducedappetite, or of reduced weight. In other methods described herein, thepatient may be desirous of having a reduced intake in food, of having areduced appetite, or of having a reduced weight. The patient may be ofany weight, and can be overweight or obese.

The compounds described herein and pharmaceutical compositionscomprising the compounds are useful for treating overweight and obesity.The methods for treating overweight provide administering to a patientin need thereof a therapeutically effective amount of one or more of theN-terminus conformationally constrained compounds or the N-terminusconformationally constrained GLP-1 receptor agonist compounds describedherein to treat overweight in the patient. The methods for treatingobesity provide administering to a patient in need thereof atherapeutically effective amount of one or more of the N-terminusconformationally constrained compounds or the N-terminusconformationally constrained GLP-1 receptor agonist compounds describedherein to treat obesity in the patient.

The disclosure also provides drug delivery devices having at least onetherapeutically effective dose of the compounds described herein or thepharmaceutical composition containing the compounds described herein.The drug delivery devices can be single or multiple-use vials, single ormultiple-use pharmaceutical pens, single or multiple-use cartridges, andthe like. In one embodiment, the drug delivery devices contain thecompounds or pharmaceutical compositions described herein in amountscapable of providing a patient with from about 7 to about 40 doses orenough doses to last about one week or about one month.

EXAMPLES

The following examples are for purposes of illustration only and are notintended to limit the scope of the claims.

Example 1 Preparation of Compound in FIG. 16A

A calculated 100 μmol of Fmoc-Ser(OtBu)-Wang resin was weighed into areaction vessel in a Symphony peptide synthesizer, and the peptideelongation was carried out following standard Fmoc peptide synthesisprotocol up to residue Thr⁵. The resulting peptide-resin intermediate(0.3 g, 0.0927 mmol) was swollen in dimethylformamide (DMF) and to theslurry was added compound 7 (0.110 g, 2.2 eq), followed byO-(benzotriazol-1-yl)-N,N,N′,N′ tetramethyluronium hexafluorophosphate(HBTU) (0.035 g, 2.2 eq), N-hydroxybenzotriazole (HOBt) (0.03 g, 2.2 eq)and methylmorpholine (NMM) (0.04 mL, 4.4 eq). After 3 hours, the resinwas washed with DMF 6×, treated with 20% piperidine in DMF 2×25 min andwashed with DMF 6×. The above cycle was repeated with Fmoc-Ala-OH andFmoc-His(Trt)-OH followed by cleavage of the peptide from the resin with10 ml TFA/H₂O/PhOH/TIPS (95:2:2:1) (TFA is trifluoroacetic acid; TIPS istriisopropylsilyl), precipitated by methyl-tert-Butyl ether and theobtained residue applied to a reverse-phase high performance liquidchromatography (HPLC) column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30min gradient) to afford the titled compound as a white powder (16.4 mg,6%): Retention time in reverse phase-high performance liquidchromatography (RP-HPLC) (C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min)is 9.78 min; Calculated mass for C₁₈₉H₂₉₄N₄₈O₆₀S (M+H)⁺4231.84. found byliquid chromatography/mass spectrometry (LC-MS) 1411.3 (M+3H)³⁺, 1059.6(M+4H)⁴⁺, 2117.2 (M+2H)²⁺.

Example 2 Preparation of Compound in FIG. 16B

A calculated 100 μmol of Fmoc-Ser(OtBu)-Wang resin was weighed into areaction vessel in a Symphony peptide synthesizer, and the peptideelongation was carried out following standard Fmoc peptide synthesisprotocol up to residue Thr⁵. The resulting peptide-resin intermediate(0.3 g, 0.0927 mmol) was swollen in DMF and to the slurry was addedcompound 24 (0.122 g, 2.2 eq), followed by HBTU (0.035 g, 2.2 eq), HOBt(0.03 g, 2.2 eq) and NMM (0.04 mL, 4.4 eq). After 3 hours, the resin waswashed with DMF 6×, treated with 20% piperidine in DMF 2×25 min andwashed with DMF 6×. The above cycle was repeated with Fmoc-His(Trt)-OHfollowed by cleavage of the peptide from the resin with 10 mlTFA/H₂O/PhOH/TIPS (95:2:2:1), precipitated by methyl-tert-Butyl ether.The obtained crude peptide was dissolved in 1.5 mL of MeOH:ACN (1:1)(ACN is acetonitrile), followed by addition of 2M LiOH (0.6 mL) and thereaction stirred at RT for 6 h. The crude was dissolved and applied to areverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 mingradient) to afford the titled compound as a white powder (15 mg, 6%):Retention time in RP-HPLC(C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min)is 9.82 min; Calculated mass for C₁₈₈H₂₉₄N₄₈O₆₀ (M+H)⁺ 4186.72. found byLC-MS 1396.7 (M+3H)³⁺, 1048.6 (M+4H)⁴⁺, 2114.2 (M+2H)²⁺.

Example 3 Preparation of Compound in FIG. 16C

A calculated 100 μmol of Fmoc-Ser(OtBu)-Wang resin was weighed into areaction vessel in a Symphony peptide synthesizer, and the peptideelongation was carried out following standard Fmoc peptide synthesisprotocol up to residue Thr⁵. The resulting peptide-resin intermediate(0.3 g, 0.0927 mmol) was swollen in DMF and to the slurry was addedcompound 18 (0.131 g, 2.2 eq), followed by HBTU (0.035 g, 2.2 eq), HOBt(0.03 g, 2.2 eq) and NMM (0.04 mL, 4.4 eq). After 3 hours, the resin waswashed with DMF 6×, treated with 20% piperidine in DMF 2×25 min andwashed with DMF 6×. The above cycle was repeated with Fmoc-His(Trt)-OHfollowed by cleavage of the peptide from the resin with 10 mlTFA/H₂O/PhOH/TIPS (95:2:2:1), precipitated by methyl-tert-Butyl ether.The obtained crude peptide was dissolved in 1.5 mL of MeOH:ACN (1:1),followed by addition of 2M LiOH (0.6 mL) and the reaction stirred at RTfor 6 h. The crude was dissolved and applied to a reverse-phase HPLCcolumn (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) toafford the titled compound as a white powder (19.7 mg, 8%): Retentiontime in RP-HPLC(C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 8.58min; Calculated mass for C₁₈₉H₂₉₄N₄₈O₆₀S (M+H)⁺ 4230.80. found by LC-MS1411. (M+3H)³⁺, 1059.6 (M+4H)⁴⁺, 2117.2 (M+2H)²⁺.

Example 4 Preparation of Compound in FIG. 16E

The synthesis of this compound was accomplished following the sameexperimental procedure as described for Example 3. The only differencewas the sequence of the peptide-resin intermediate, starting withRink-amide resin. The crude was dissolved and applied to a reverse-phaseHPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) toafford the titled compound as a white powder (15.7 mg, 7%): Retentiontime in RP-HPLC(C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 8.25min; Calculated mass for C₁₈₈H₂₈₆N₄₈O₆₀S (M+H)⁺ 4238.74. found by LC-MS1414.8 (M+3H)³⁺, 1061.6 (M+4H)⁴⁺, 2121.2 (M+2H)²⁺.

Example 5 Preparation of Compound in FIG. 16D

A calculated 100 μmol of Fmoc-Ser(OtBu)-Wang resin was weighed into areaction vessel in a Symphony peptide synthesizer, and the peptideelongation was carried out following standard Fmoc peptide synthesisprotocol up to residue Thr⁵. The resulting peptide-resin intermediate(0.3 g, 0.0927 mmol) was swollen in DMF and to the slurry was addedcompound 11 (0.100 g, 2.2 eq), followed by HBTU (0.035 g, 2.2 eq), HOBt(0.03 g, 2.2 eq) and NMM (0.04 mL, 4.4 eq). After 3 h, the resin waswashed with DMF 6×, treated with 20% piperidine in DMF 2×25 min andwashed with DMF 6×. The above cycle was repeated with Fmoc-Ala-OH andFmoc-His(Trt)-OH followed by cleavage of the peptide from the resin with10 ml TFA/H₂O/PhOH/TIPS (95:2:2:1), precipitated by methyl-tert-Butylether and the obtained residue applied to a reverse-phase HPLC column(C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford thetitled compound as a white powder (20.3 mg, 10%): Retention time inRP-HPLC(C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 9.74 min;Calculated mass for C₁₈₈H₂₉₄N₄₈O₆₀ (M+H)⁺ 4186.72. found by LC-MS 1396.7(M+3H)³⁺, 1048.6 (M+4H)⁴⁺, 2114.2 (M+2H)²⁺.

Example 6 Preparation of Compound in FIG. 16F

A calculated 100 μmol of Fmoc-Ser(OtBu)-Wang resin was weighed into areaction vessel in a Symphony peptide synthesizer, and the peptideelongation was carried out following standard Fmoc peptide synthesisprotocol up to residue Thr⁵. The resulting peptide-resin intermediate(0.3 g, 0.0927 mmol) was swollen in DMF and to the slurry was addedcompound 21 (0.125 g, 2.2 eq), followed by HBTU (0.035 g, 2.2 eq), HOBt(0.03 g, 2.2 eq) and NMM (0.04 mL, 4.4 eq). After 3 h, the resin waswashed with DMF 6×, treated with 20% piperidine in DMF 2×25 min andwashed with DMF 6×. The above cycle was repeated with Fmoc-His(Trt)-OHfollowed by cleavage of the peptide from the resin with 10 mlTFA/H₂O/PhOH/TIPS (95:2:2:1), precipitated by methyl-tert-Butyl ether.The obtained crude peptide was dissolved in 1.5 mL of MeOH:ACN (1:1),followed by addition of 2M LiOH (0.6 mL) and the reaction stirred at RTfor 6 h. The crude was dissolved and applied to a reverse-phase HPLCcolumn (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) toafford the titled compound as a white powder (8 mg, 4%): Retention timein RP-HPLC(C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 7.93 min;Calculated mass for C₁₈₉H₂₉₆N₄₈O₆₀ (M+H)⁺ 4200.75. found by LC-MS 1401.8(M+3H)³⁺, 1051.6 (M+4H)⁴⁺, 2102.2 (M+2H)²⁺.

Example 7 Preparation of Compound in FIG. 16G

The synthesis of this compound was accomplished following the sameexperimental procedure as described for Example 3. The only differencewas the sequence of the peptide-resin intermediate. The crude wasdissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CNin 0.1% TFA/H₂O over 30 min gradient) to afford the titled compound as awhite powder (27.4 mg, 18%): Retention time in RP-HPLC(C₁₈, 5-75% CH₃CNin 0.1% TFA/H₂O over 15 min) is 8.25 min; Calculated mass forC₁₅₃H₂₂₈N₄₀O₄₇S (M+H)⁺ 3411.83. found by LC-MS 1138.6 (M+3H)³⁺, 854.5(M+4H)⁴⁺, 1707.9 (M+2H)²⁺.

Example 8 Preparation of (5,5)-Glu-Gly-OH as shown in FIG. 18

Preparation of Boc-Asp(OBn)-OMe: Boc-Asp(OBn)-OH 2.4 g (1 equiv, 7.45mmol) was dissolved in 17 mL of dry DMF in a 100 mL round-bottom flask.Finely ground K₂CO₃ (1.5 g, 11 mmol) was added to the solution to form asuspension. The mixture was cooled to 0° C. in an ice-bath over fiveminutes. MeI (1 mL, 15 mmol) was then added to the mixture over 20seconds under a positive flow of nitrogen. A yellow color developedwithin 30 min. The resulting mixture was stirred for 3 hours. Theice-bath was removed and 25 mL of water were added and the mixture leftstanding at room temperature 2 hours. The mixture was then treated with30 mL of water and extracted with 3×25 mL AcOEt. The organics werewashed with saturated NaHCO₃ 1×, brine 3×, dried over Na₂SO₄ filteredand concentrated to yield a yellow oil. This material was passed througha short silica column (eluting w/ AcOEt), after concentration of thepure fractions 2.42 g of Boc-Asp(OBn)-OMe as a yellow oil was obtained(98% yield). ¹H NMR (DMSO d6, 500 mHz): δ 7.36 (m, 5H), 5.10 (s, 2H),4.40 (q, 1H), 3.6 (s, 3H), 2.80 (dd, 1H), 2.72 (dd, 1H), 1.38 (s, 9H).LCMS (C₁₈, 2-98% CH₃CN in 0.1% TFA/H₂O over 6 min); Calculated mass forC₁₇H₂₃NO₆ (M+H)⁺ 338.23. found by LC-MS 338.2.

Preparation of Compound 1 in FIG. 18: Boc-Asp(OBn)-OMe (26.4 g, 78.3mmol) was dissolved in a 500 mL round-bottom flask with 110 mL of dryTHF and the mixture cooled at negative 42° C. To this mixture was addedlithium bis(trimethylsilyl)amide (LiHMDS) (173 mL, 1 M solution, 2.2eq). This solution was stirred under a gentle argon flow for 45 minfollowed by slow addition (via syringe) of tert-butyl bromo acetate(14.5 mL, 1.25 eq). Thin layer chromatography (TLC) (Hex:AcOEt, 8/2)showed after 4 h>95% of expected product. The reaction was quenched byaddition of saturated NH₄Cl (70 mL). The mixture was evaporated and theresidue re-dissolved with 100 mL of dichloromethane (DCM). The emulsionformed was separated by standing overnight. The organics were collectedand washed with saturated NH₄Cl 2×, brine 1×, dried over Na₂SO₄,filtered and concentrated to give Compound 1 as a red orange oil. Flashchromatography (Hex:AcOEt, 7:3) gave 27.4 g of a yellow oil (78% yield).¹H NMR (DMSO d6, 500 mHz): δ 7.35 (m, 5H), 5.08 (s, 2H), 4.57 (dd, 1H),4.05 (m, 1H), 3.52 (s, 3H), 1.39 (s, 9H), 1.34 (s, 9H). LCMS (C₁₈, 2-98%CH₃CN in 0.1% TFA/H₂O over 6 min); Calculated mass for C₂₃H₃₃NO₈ (M+H)⁺452.23. found by LC-MS 452.2.

Preparation of Compound 2 in FIG. 18. Compound 2 istert-butyl-(1S,2R)-2-[(carboxy)-3-tert-butoxycarbonyl)-1-metoxhycarbonyl)]propylcarbamate.Compound 1 (5.8 g, 12.8 mmol) was dissolved in 30 mL of a 6:4 mixture ofMeOH/THF. Argon was bubbled in the catalyst for 5 minutes, followed byincorporation of two hydrogen balloons attached via syringe to thereaction flask. After 4 hours, LCMS shows complete transformation. Thecrude mixture was filtrated through celite and concentrated to yield5.25 g of Compound 2 as an orange oil which was used without furtherpurification in the next step. Calculated mass for C₁₆H₂₇NO₈ (M+H)⁺362.23. found by LC-MS 362.2.

Preparation of Compound 3 in FIG. 18. Compound 3 istert-butyl-(1S,2R)-2-[(benzylthio)-carbonyl)-3-tert-butoxycarbonyl)-1-metoxhycarbonyl)]propylcarbamate.Compound 2 (2.1 g, 5.8 mmol) was dissolved with DCM (7 mL). To thisclear mixture was added ethylthiol (0.4 g, 1.1 eq) and4-dimethylaminopyridine (DMAP) (0.07 g, 0.1 eq). To the clear solutionwas added dicyclohexyl carbodiimide (DCC) (1.3 g, 1.1 eq) as a solid andthe mixture was stirred at room temperature for 5 hours. The mixture wasdiluted with DCM (40 mL) and washed with 1N HCl (2×), dried over Na₂SO₄,filtered and concentrated to give 2.15 g of Compound 3 as a yellow oil,which was used without further purification in the next step. Calculatedmass for C₁₈H₃₁NO₇S (M+H)⁺ 406.13. found by LC-MS 406.2.

Preparation of Compound 4 in FIG. 18. Compound 4 istert-butyl-(1S,2R)-3-(tert-butoxcarbonyl)-1-(methoxycarbonyl)-2-formylpropylcarbamate.To Compound 3 (5.9 g, 14.5 mmol) and Pd—C 10% wt (0.35 g) were addedacetone (36 mL) and the mixture cooled down to about 4-8° C. To thismixture was added drop wise, under positive flow of Argon, triethylsilyl(TES) (11.5 mL, 5 eq). After addition of TES the mixture was kept at10-15° C. After 3.5 hours LCMS showed no more starting material. Themixture was filtrated through celite, and the solution concentrated togive 6.6 g of Compound 4 as a green oil which was used without furtherpurification in the next step. Calculated mass for C₁₆H₂₇NO₇ (M+H)⁺346.13. found by LC-MS 346.2.

Preparation of Compound 5 in FIG. 18. Compound 5 ismethyl-[3S,4R,8R]-1-Aza-3-tert-butoxycarbonyl-4-(tert-butoxycarbonyl)methyl)-2-oxo-6-thiabicyclic[3.3.0]-octane-8-carboxylate.To Compound 4 (1.3 g, 3.76 mmol), L-Cys-OH—HCl (0.98 g, 1.5 eq) and 4° Amolecular sieves (2g) was added dry pyridine (10 mL) and the mixturestirred, under argon, at room temperature for 4 hours in high pressurevessel. After this time, 3.5 mL more of pyridine were added and thereaction was stirred at 50° C. for 5 days. After this time, the crudemixture was filtered through celite, the solution was re-dissolved inAcOEt and washed with 2N HCl 2×, dried over Na₂SO₄, filtered andconcentrated to yield 1.2 g of Compound 5 as a yellowish semisolid,which was used without further purification in the next step. Calculatedmass for C₁₉H₃₀N₂O₇S (M+H)⁺ 431.13. found by LC-MS 431.2.

Preparation of Compound 6 in FIG. 18. Compound 6 is[3S,4R,8R]-1-Aza-3-amino-4-(tert-butoxycarbonyl)methyl)-2-oxo-6-thiabicyclic[3.3.0]-octane-8-carboxylate.Crude Compound 5 was treated with 10 mL of a 50% TFA-DCM mixture at 0°C. The mixture was let to warm up at 10° C. and stirred for 2.5 hours.The mixture is then concentrated and the residue re-dissolved with 5 mLof a 2M LiOH solution and stirred at room temperature for 2.5 hours.LCMS analysis showed complete transformation to Compound 6. The reactionwas concentrated and the residue passed through a short column packedwith ion-exchange resin (H⁺, Dowex), eluting with 1:1 MeOH/H₂O. Theresidue was concentrated and then lyophilized to give 1.8 g of crudeCompound 6 as a yellow semisolid, which was used without furtherpurification in the next step. Calculated mass for C₁₃H₂₀N₂O₅S (M+H)⁺317.13. found by LC-MS 317.2.

Preparation of Compound 7 in FIG. 18. Compound 7 is the (5,5)-Glu-Glybicylic dipeptide mimetic. Compound 7 is also referred to as[3S,4R,8R]-1-Aza-3-fluorenylmethyl-carbonyl-4-(tert-butoxycarbonyl)methyl)-2-oxo-6-thiabicyclic[3.3.0]-octane-8-carboxylate.Crude Compound 6 (1.2 g, 3.8 mmol) was dissolved in 17 mL of dry DCM. Tothis solution was added FmocOSu (1.8 g, 1.4 eq) followed byN,N-diisopropylethylamine (DIEA) (1.3 mL, 2 eq). The reaction wasstirred at room temperature for 3 hours. To the mixture was added 50 mLof DCM and washed with 2M HCl 2×, brine 1×, dried over Na₂SO₄, filteredand concentrated to give 1.42 g of a yellow oil. Purification by flashchromatography using an increasing polarity solvent gradient (Hex:AcOEt1:1 to DCM:MeOH 9:1) gave 0.150 g of pure Compound 7. Calculated massfor C₂₈H₃₀N₂O₇S (M+H)⁺ 539.13. found by LC-MS 539.2.

Example 9 Preparation of Compound 11 in FIG. 19

Compound 9 is1-Aza-3-amino-tert-butoxylcarbonyl-4-(tert-butoxycarbonyl)methyl)-2-oxo-1-methoxyacetyl.Compound 4 was prepared as described above. A solution of Compound 4(preparation described in Example 8) (4.1 g, 12 mmol) and the HCl saltof NH₂-Gly-OMe (1.66 g, 1.1 eq) in 20 mL of DMF were stirred at roomtemperature. To this mixture was added NaBH(OAc)₃ (5.0 g, 2 eq)dissolved in 18 mL of DMF. After 1 hour LCMS shows completetransformation to the desired secondary amine 8. Calculated mass forC₁₉H₃₄N₂O₈ (M+H)⁺ 419.1. found by LC-MS 419.2. To this crude mixture wasadded AcOEt (80 mL), washed with sat. NaHCO₃ 3×, water 1×, brine 1×,dried over Na₂SO₄, filtered and concentrated to give 3.9 g of a crudeyellow oil. LCMS of this material showed that the five-member ringlactam (Compound 9) was formed during work-up. The residue wasconcentrated to give 3.9 g of crude Compound 9 as a yellow oil, whichwas used without further purification in the next step. Calculated massfor C₁₈H₃₀N₂O₇ (M+H)⁺ 387.1. found by LC-MS 387.2.

Preparation of Compound 10 in FIG. 19. Compound 10 is1-amino-4-(tert-butoxy-carbonyl)methyl)-2-oxo-1-methoxyacetyl. CrudeCompound 9 (3.8 g) was cooled at 10° C. in an ice-water bath. To thisstirred mixture was added in a drop wise manner 15 mL of a 50% solutionof TFA in DCM. The mixture was stirred at that temperature for 2 h. LCMSshowed a selective N-Boc deprotection. The mixture was concentrated toyield crude compound 10 as clear semisolid, which was used withoutfurther purification in the next step. Calculated mass for C₁₃H₂₂N₂O₅(M+H)⁺ 287.1. found by LC-MS 287.2.

Preparation of Compound 11 in FIG. 19. Compound 11 is a γ-lactam-Glu-Glybicyclic dipeptide mimetic which is1-Aza-3-aminofluorenylmethylcarbonyl-4-(tert-butoxycarbonyl)methyl)-2-oxo-1-acetylcarboxylate.Crude Compound 10 (2.8 g, 9.8 mmol) was dissolved in 8 mL of a 1:1mixture of dioxane/MeOH at room temperature. To this mixture was added15 mL of a 2M LiOH (2.8 eq) and the mixture stirred at room temperaturefor 3 hours. The mixture was then concentrated and passed trough a shortcolumn of Dowex H⁺ ion-exchange resin. The pooled fractions containingM+1=273 by LCMS were collected and lyophilized. This crude material wasthen dissolved in DCM (40 mL) followed by addition of DIEA (3.5 mL, 2eq) and FmocOSu (3.9 g, 1.2 eq). The mixture was stirred at roomtemperature for 3 hours. LCMS analysis shows no more starting material,thus to the reaction was added AcOEt (60 mL), washed with sat. NaHCO₃3×, water 1×, brine 1×, dried over Na₂SO₄, filtered and concentrated togive 2.5 g of a crude Compound 11. The mixture was purified by flashchromatography using AcOEt:Hex (1:1). Collection of the pure fractionsidentified by LCMS gave 100 mg of pure Compound 11. Calculated mass forC₂₇H₃₀N₂O₇ (M+H)⁺ 495.1. found by LC-MS 495.2.

Example 10 Preparation of Compound 18 in FIG. 20

Compound 1 (i.e.,tert-butyl-1(1S,2R)-2-[(benzyloxy)carbonyl)-3-tert-butoxycarbonyl)-1-metoxhycarbonyl)]propylcarbamate)was prepared as described in Example 8 above.

Preparation of Compound 12 in FIG. 20. Compound 12 istert-Butyl-(1S,2R)-2-[(benzylcarboxylate)-3-carboxy)-1-metoxhycarbonyl)]propylcarbamate.To Compound 1 (13.9 g, 30.8 mmol) was added 45 mL of a 2M solution ofHCl in diethyl ether. The clear yellow solution was stirred at roomtemperature for 18 hours. The mixture was triturated with cold etherwhich gave a yellow foam. Drying of this solid gave 8.32 g (84% yield)of the hydrochloride salt intermediate which was used without furtherpurification in the next step. This HCl salt crude (8.3 g, 25 mmol) wasdissolved in THF (80 mL). To this clear solution was added TEA (7.6 mL,2.2 eq) followed by Boc₂O (6.5 g, 1.2 eq). The mixture was stirred for18 hours at room temperature. The mixture was then concentrated,re-dissolved in AcOEt (150 mL), washed with 1N HCl 2×, sat. NaCl 1×,dried over Na₂SO₄, filtered, concentrated and dried under high vacuum toyield Compound 12 as a brownish oil (11.1 g, 100% crude yield), whichwas used without further purification in the next step. Calculated massfor C₁₉H₂₅NO₈ (M+H)⁺ 396.1. found by LC-MS 396.2.

Preparation of Compound 13 in FIG. 20. Compound 13 istert-Butyl-(1S,2R)-2-[(benzylcarboxylate)-3-ethylthiocarboxylate)-1-metoxhycarbonyl)]propylcarbamate.DCC (5.9 g, 1.1 eq) was added to a solution of crude Compound 12 (10.4g, 26.2 mmol), EtSH (2.15 mL, 1.1 eq) and 4-dimethylaminopyridine (DMAP)(0.32 g, 0.1 eq) dissolved in DCM (28 mL). After 6 hours the mixture wasconcentrated, re-dissolved in AcOEt (150 mL) washed with 1N HCl 2×, sat.NaCl 1×, dried over Na₂SO₄, filtered, concentrated and dried under highvacuum to yield Compound 13 as a brown oil (7.6 g), which was usedwithout further purification in the next step. Calculated mass forC₂₁H₂₉NO₇S (M+H)⁺ 440.1. found by LC-MS 440.2.

Preparation of Compound 14 in FIG. 20. Compound 14 is(1S,2R)-2-[(benzyl-carboxylate)-3-ethylthiocarboxylate)-1-metoxhycarbonyl)]propylaminehydrochloride salt. Crude Compound 13 (7.6 g, 17.2 mmol) was dissolvedin ethyl ether (8 mL), followed by addition of a 2N solution of HCl indiethyl ether (40 mL). The clear mixture was stirred at room temperaturefor 18 hours. The mixture was concentrated to give Compound 14 as anorange oil (7.5 g) which was used without further purification in thenext step. Calculated mass for C₁₆H₂₁NO₅S (M+H)⁺ 338.1. found by LC-MS338.2.

Preparation of Compound 15 in FIG. 20. Compound 15 is(1S,2R)-2-[(benzyl-carboxylate)-3-ethylthiocarboxylate)-1-metoxhycarbonyl)]propyl-L-N-Boc-Ala.Crude Compound 14 (6.6 g, 17.7 mmol), Boc-L-Ala-OH (3.7 g, 1.1 eq) and2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoride(HATU) (12.1 g, 1.8 eq) were dissolved in ACN (150 mL). To this solutionwas added DIEA (6.1 mL, 2 eq) and the mixture was stirred at roomtemperature for 6 hours. The mixture was concentrated, re-dissolved inAcOEt (150 mL), washed with sat NH₄Cl 2×, sat. NaCl 1×, dried overNa₂SO₄, filtered, concentrated and dried under high vacuum to yieldcompound 15 as a brown oil (16.5 g of crude). Purification by flashchromatography using Hex;AcOEt (6:4) gave pure compound 15 (3.55 g, 40%yield). Calculated mass for C₂₄H₃₄N₂O₈S (M+H)⁺ 511.1. found by LC-MS511.2.

Preparation of Compound 16 in FIG. 20. Compound 16 is(1S,2R)-2-[(benzyl-carboxylate)-3-formyl)-1-metoxhycarbonyl)]propyl-L-N-Boc-Ala.Triethylsilane (5.6 mL, 5 eq) was slowly added, under argon, to asolution of Compound 15 (3.5 g, 6.9 mmol) and Pd—C (0.6 g) dissolved inacetone (16 mL) in an ice bath (˜4° C.). After addition of TES thereaction was stirred at 10-20° C. After 4 hours, thin layerchromatography (TLC) showed complete transformation. The reaction wasfiltered and concentrated to give an off-green oil. Purification byflash chromatography using Hex;AcOEt (6:4) gave pure Compound 16 (2.9 g,93% yield). Calculated mass for C₂₂H₃₀N₂O₈ (M+H)⁺ 452.1. found by LC-MS452.2.

Preparation of Compound 17 in FIG. 20. Compound 17 is(3R,6S,7R)-7-(benzyloxy-carbonyl)-6-amino-(L-Boc-Ala)-hexahydro-5-oxo-2H-thiazolo[3,2-a]pyridine-3-carboxylicacid. In a high pressure vial crude Compound 16 (2 g, 4.55 mmol) wasdissolved in dry pyridine (12 mL). To this mixture was added L-Cys-OH(0.88 g, 1.6 eq) followed by addition of 2.7 g of activated 4° Amolecular sieves (powder). The mixture was stirred vigorously at roomtemperature for 4 hours. Dry-pyridine (10 mL) was added to the mixture,the vial was capped and the mixture stirred at 50° C. for 4 days. Themixture was then filtered through a bed of celite and washed withtetrahydrofuran (THF) and MeOH. The mixture was concentrated to giveCompound 17 as yellow foam (1.6 g) which was used without furtherpurification in the next step. Calculated mass for C₂₄H₃₁N₃O₈S (M+H)⁺522.1. found by LC-MS 522.2.

Preparation of Compound 18 in FIG. 20. Compound 18 is(3R,6S,7R)-7-(benzyloxy-carbonyl)-6-amino-(L-Ala)-hexahydro-5-oxo-2H-thiazolo[3,2-a]pyridine-3-carboxylicacid. In a round bottom flask crude Compound 17 (1.6 g, 3.1 mmol) wasdissolved in dioxane (15 mL) followed by addition of 4M HCl. The mixturewas stirred at room temperature and after 3 hours LCMS analysis showedcomplete conversion to the free amine. The mixture was concentrated,dissolved with DCM, MeOH, concentrated again and then dried under highvacuum to give the hydrochloride salt as a brownish semisolid (1.5 g)which was used without further purification in the next step. Calculatedmass for C₂₄H₃₁N₃O₈S (M+H)⁺ 522.1. found by LC-MS 522.2.

This HCl salt was dissolved in DCM (14 mL) followed by addition of DIEA(2 mL, 3.5 eq) and FmocOSu (1.2 g, 1.1 eq) in portions as solid. Themixture was stirred for 3 hours showing complete transformation by LCMS.The mixture was concentrated, dissolved in AcOEt (60 mL), washed with 2NHCl 2×, brine, dried over Na₂SO₄, filtered and concentrated to give 2.1g of a brownish oil. Purification by flash chromatography usingHex;AcOEt (5:95) followed by 100% MeOH gave pure compound 18 (0.821 g,44% yield). Calculated mass for C₃₄H₃₃N₃O₈S (M+H)⁺ 644.1. found by LC-MS644.2.

Example 11 Preparation of Compounds in FIG. 21

Preparation of compound 16 has been described in Example 10.

Preparation of Compound 19 in FIG. 21. Compound 19 is(1S,2R)-[2-(benzylcarboxylate)-3-formyl-4-(tert-butoxy-N-2-methylacetyl)-1-(methoxycarbonyl)]butyl-L-N-Boc-Ala. Crude compound 16 (1.01g, 2.24 mmol) and L-Ala-OtBu. HCl (0.449 g, 1.1 eq) were dissolved inDMF (4.5 mL) at room temperature. NaBH(OAc)₃ (0.95 g, 2 eq) wasdissolved separately in DMF (4.5 mL) at room temperature, the twosolutions were mixed and stirred at room temperature for about 2 hours.The reaction was washed with saturated NaHCO₃ solution 2×, water 1×,sat. NaCl 1×, dried over Na₂SO₄, filtered, concentrated to yield 0.92 gof Compound 19 as a colorless oil, which was used without furtherpurification in the next step. Calculated mass for C₂₉H₄₅N₃O₉ (M+H)⁺579.68. found by LC-MS 579.6.

Preparation of Compound 20 in FIG. 21. Compound 20 is(1S,2R)-tert-butyl-[4-(benzylcarboxylate)-3-(N-L-Boc-Ala)-2-oxopiperidine)-1-methyl-1-yl]acetate.Crude Compound 19 (0.550 g, 0.95 mmol) was dissolved in DMF (3 mL), DIEA(0.445 mL, 2 eq) was heated using microwaves at 140° C. for 20 min. Thereaction was checked and then washed with 0.5M HCl 2×, water 1×, sat.NaCl 1×, dried over Na₂SO₄, filtered, concentrated to give 0.50 g ofCompound 20 as a light brownish oil, which was used without furtherpurification in the next step. Calculated mass for C₂₈H₄₁N₃O₈ (M+H)⁺547.64. found by LC-MS 547.6.

Preparation of Compound 21 in FIG. 21. Compound 21 is(1S,2R)-[4-(benzyl-carboxylate)-3-(N-L-Fmoc-Ala)-2-oxopiperidine)-1-methyl-1-yl]aceticacid. Crude Compound 20 (0.501 g, 0.91 mmol) was dissolved in 5 mL of20% TFA in DCM and stirred for about an hour at room temperature. Thereaction was concentrated and then dried under high vacuum to give thecorresponding TFA salt as brownish oil (0.47 g) which was used withoutfurther purification in the next step. Calculated mass for C₁₉H₂₅N₃O₆(M+H)⁺ 505.64. found by LC-MS 505.6

This TFA salt (0.47 g, 0.91 mmol) was dissolved in aqueous 10% Na₂CO₃solution (12 mL) and DMF (4 mL), and cooled down to 0° C. A solution ofFmocOSu (0.472 g, 1.5 eq) in DMF (8 mL) was added dropwise to the coldaqueous solution. After 5 minutes the ice bath was removed and thereaction was stirred overnight, quenched by adding 40 mL water followedby washings with AcOEt. The aqueous layer was acidified using 2N HCl(pH=2) and washed with AcOEt 3×, NaCl 1×, dried over Na₂SO₄, filteredand concentrated. Purification was done by washing the crude compoundwith Hex:AcOEt (6:4) 2× at room temperature to give Compound 21 as lightcolored oil (0.290 g, 52% yield). Calculated mass for C₃₄H₃₅N₃O₈ (M+H)⁺613.24. found by LC-MS 613.2.

Preparation of Compound 22 in FIG. 21. Compound 22 is(1S,2R)-[2-(benzyl-carboxylate)-3-(formyl)-4-(tert-butoxy-N-acetyl)-1-(methoxycarbonyl)]butyl-L-N-Boc-Ala.Crude compound 16 (1.01 g, 2.24 mmol) and AcOH. NH₂-Gly-OtBu (0.478 g,1.1 eq) were dissolved in dry DMF (4.5 mL) at room temperature.NaBH(OAc)₃ (0.95 g, 2 eq) was dissolved separately in dry DMF (4.5 mL)at room temperature, the two solutions were mixed and stirred at roomtemperature for about 2 hours. The reaction was washed with saturatedNaHCO₃ solution 2×, water 1×, sat. NaCl 1×, dried over Na₂SO₄, filteredand concentrated to yield 1.09 g of Compound 22 as a colorless oil,which was used without further purification in the next step. Calculatedmass for C₂₈H₄₃N₃O₉ (M+H)⁺ 565.68. found by LC-MS 565.3.

Preparation of Compound 23 in FIG. 21. Compound 23 is(1S,2R)-tert-butyl-[4-(benzylcarboxylate)-3-(N-L-Boc-Ala)-2-oxopiperidine)-1-yl]acetate.Crude compound 22 (0.550 g, 0.95 mmol) was dissolved in DMF (3 mL), DIEA(0.445 mL, 2 eq) and then heated using microwaves at 140° C. for 20 min(Biotage™, Initiator8). The reaction was checked by LCMS. The reactioncrude was washed with 0.5M HCl 2×, water 1×, sat. NaCl 1×, dried overNa₂SO₄, filtered and concentrated to yield 0.41 g of Compound 23 as alight brownish oil, which was used without further purification in thenext step. Calculated mass for C₂₇H₃₉N₃O₈ (M+H)⁺ 533.64. found by LC-MS533.6.

Preparation of Compound 24 in FIG. 21. Compound 24 is(1S,2R)-[4-(benzyl-carboxylate)-3-(N-L-Fmoc-Ala)-2-oxopiperidine)-1-yl]aceticacid. Crude Compound 23 (0.41 g, 0.76 mmol) was dissolved in 5 mL of 20%TFA in DCM; reaction was stirred for about an hour at room temperature.The reaction was concentrated and then dried under high vacuum to givethe corresponding TFA salt as a brownish oil (0.45 g) which was usedwithout further purification in the next step. Calculated mass forC₁₈H₂₃N₃O₆ (M+H)⁺ 491.3. found by LC-MS 491.4

This TFA salt (0.45 g, 0.92 mmol) was dissolved in aqueous 10% Na₂CO₃solution (12 mL) and DMF (4 mL), and cooled down to 0° C. A solution ofFmocOSu (0.472 g, 1.5 eq) in DMF (8 mL) was added dropwise to the coldaqueous solution. After 5 min, the ice bath was removed and the reactionwas stirred overnight, then quenched by adding about 40 mL water,followed by washings with AcOEt. The aqueous layer was acidified using2N HCl (pH=2) and washed with AcOEt 3×, NaCl 1×, dried over Na₂SO₄,filtered, concentrated. Purification was done by washing the crudecompound with Hex:AcOEt (6:4) 2× at room temperature giving Compound 24as light colored oil (0.15 g, 27% yield). Calculated mass for C₃₃H₃₃N₃O₈(M+H)⁺ 599.6. found by LC-MS 599.4.

Example 12 Preparation of Compounds in FIGS. 1A, 2A, and 5A

The compound in FIG. 1A may be prepared following the methods described,e.g., in U.S. Pat. No. 6,872,700, the disclosure of which isincorporated by reference. The compounds in FIGS. 2A and 5A may beprepared following the methods described, e.g., in WO 2007/139941, thedisclosure of which is incorporated by reference.

Example 13 Preparation of Compounds in FIGS. 1B-C, 2B-U, 3A-G, 5B-G,6A-E, 10A-I, 11A-C, 12, 13A-B, 14B-C, 14E-R

For each compound in FIGS. 1B, 1C, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I, 2J,2K, 3A, 3B, 3C, 3D, 3E, 3F, 3G, 5B, 5C, 5D, 6A, 6B, 6C, 6D, and 6E, acalculated 100 μmol of Fmoc-Ser(OtBu)-Wang resin or Fmoc Rink amideresin was weighed into a reaction vessel in a Symphony peptidesynthesizer, and peptide elongation was carried out following standardFmoc peptide synthesis protocol. If an unnatural amino acid was used atposition 2 this was incorporated manually in a polypropylene syringe.Cleavage of the peptide from the resin was done with 10 mLTFA/H₂O/PhOH/TIPS (95:2:2:1), then precipitated by methyl-tert-Butylether. The crude was dissolved and applied to a reverse-phase HPLCcolumn (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) toafford the titled compounds as white powders. All characterized byLC-MS. The compounds in FIGS. 2L-U, 5E-G, 10A-I, 13B, 14B-C and 14E-Kwill be prepared following the methods described in this example.

With respect to the compounds in FIGS. 14L-R, a calculated 100 μmol ofFmoc-Rink-amide resin was weighed into a reaction vessel in a Symphonypeptide synthesizer, and peptide elongation was carried out followingstandard Fmoc peptide synthesis protocol. If an unnatural amino acid wasused at position 2 this was incorporated manually in a polypropylenesyringe. Only for the compounds in FIGS. 14N-O, this procedure wascarried out under the following microwave-assisted conditions: Amicrowave vial was loaded with the corresponding peptide-resinintermediate (0.3 g, 0.14 mmol), Fmoc-AA-OH (4 eq), PyBrop (0.32 g, 4.8eq), 2,6-lutidine (0.25 mL, 15 eq), dichloroethane (DCE) (2-3 mL) andDMF (about 0.3 mL). The vial was capped and heated with microwaves(Biotage™, Initiator8) at 100° C. for 11 min. The resin was filtered,washed with DCE and MeOH. Cleavage of the peptide from the resin wasdone with 10 mL TFA/H₂O/PhOH/TIPS (95:2:2:1), then precipitated bymethyl-tert-Butyl ether. The crude was dissolved and applied to areverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 mingradient) to afford the titled compounds as white powders. Allcharacterized by LC-MS.

Example 14 Preparation of Compounds in FIGS. 8A-H

For the compounds in FIGS. 8B, 8C, 8D, 8E, 8F, 8G, and 8H, a calculated100 mol of Fmoc-Ser(OtBu)-Wang or Fmoc-Rink-amide resin was weighed intoa reaction vessel in a Symphony peptide synthesizer, and the peptideelongation was carried out following standard Fmoc peptide synthesisprotocol. The corresponding L- or D-Cys(Trt)-OH was introduced atpositions 2 and 4. Cleavage of the peptide from the resin with 10 mLTFA/H₂O/PhOH/TIPS (95:2:2:1) then precipitated by methyl-tert-Butylether. The crude peptides were dried overnight under high vacuum.

Disulfide cyclization: Clear-OX resin (0.39 g, 3× molar excess) wasswollen on DCM for 45 min at room temperature, then washed with DCM 2×,DMF 3×, MeOH 3×, deionized water 3× and finally H₂O:ACN (1:1) 3×. Thecorresponding crude peptide (0.1 g) was dissolved in degassed 1:1 v/vsolution of 0.1M NH₄OAc buffer (pH=6.5)/ACN. The peptide solution wasthen added to the pre-swollen Clear-OX resin and the slurry was shackedat room temperature. After 2-3 hours the cyclization was complete. Theresin was washed with a small amount of ACN/H₂O (1:1) solution, thefiltrate concentrated to remove volatiles and then lyophilized. Thecrude was dissolved and applied to a reverse-phase HPLC column (C₁₈,20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titledcompounds as white powders. All characterized by LC-MS. The compound inFIG. 8A will be prepared following the methods described in thisexample.

Example 15 Preparation of Compounds in FIGS. 9A-H

For each compound in FIGS. 9A, 9B, 9C, 9D, 9E, 9G, and 9H, a calculated100 μmol of Fmoc-Rink amide resin was weighed into a reaction vessel ina Symphony peptide synthesizer, and the peptide elongation was carriedout following standard Fmoc peptide synthesis protocol. The amino acidat position 2 of all these compounds was introduced by special couplingconditions. A microwave vial was loaded with the correspondingpeptide-resin intermediate (0.3 g, 0.14 mmol), Fmoc-AA-OH (4 eq), PyBrop(0.32 g, 4.8 eq), 2,6-lutidine (0.25 mL, 15 eq), dichloroethane (DCE)(2-3 mL) and DMF (˜0.3 mL). The vial was capped and heated withmicrowaves (Biotage™, Initiator8) at 100° C. for 11 min. The resin wasfiltered, washed with DCE and MeOH. For some compounds the above processwas repeated twice. The resin is then treated in a cycle ofdeprotection, coupling with Fmoc-His(Trt)-OH, HBTU and HOBt,deprotection. Cleavage of the peptide from the resin was done with 10 mLTFA/H₂O/PhOH/TIPS (95:2:2:1), then precipitated by methyl-tert-Butylether. The crude was dissolved and applied to a reverse-phase HPLCcolumn (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) toafford the titled compounds as white powders. All characterized byLC-MS. The compound in FIG. 9F will be prepared following the methodsdescribed in this example.

Example 16 In Vitro Assay

The compounds shown in Table 1 below were analyzed in an in vitrofunctional assay at the GLP-1 receptor. The assay was conducted asfollows: Membrane fractions were prepared from confluent cultures of RINm5f cells. Compounds were serially diluted with an assay buffer, andthen added to a 96-well assay plate containing RIN m5f cell membranes inan ATP/GTP mixture. Cyclase activities were determined by measuring theproduction of cAMP induced through GLP-1 receptor activation.Quantification of cAMP production was achieved through a competitivechemiluminescence assay with a biotinylated-cAMP probe using PerkinElmer Fusion™-Alpha Microplate Analyzer (AlphaScreen™ technology). Thecompound EC₅₀ values were obtained through fitting theconcentration-response curves to a four-parameter logistic equationwithin GraphPad PRISM® software. The results of the assay are presentedin Table 1 below.

Example 17 In Vivo Assay

Some compounds shown in Table 1 below were analyzed in an in vivo basalglucose lowering assay using the following procedure: A subcutaneousinjection of either 200 μl phosphate-buffered saline (PBS) vehicle ortest article was given immediately following baseline glucose (t=0) toNIH/Swiss female mice. Tail blood glucose samples were measured at t=2and 4 hours post dose using a OneTouch® Ultra® (LifeScan, Inc., aJohnson & Johnson Company, Milpitas, Calif.). Body weight was measureddaily. Significant test sample effects were identified by ANOVA(p<0.05), followed by Dunnett's post test using GraphPad Prism version4.00 for Windows (GraphPad Software, San Diego Calif.). The results arepresented in Table 1 below.

TABLE 1 Cyclase GLP-1 In Vivo Compound in Receptor Glucose-LoweringFigure EC₅₀ (nM) Assay  1A 0.01 not tested  1B 0.004 not tested  1C0.029 not tested  2A 0.008 greater than 3 hours  2B 0.0089 more than 4hours  2C 6 not tested  2D 0.04 not tested  2E 2.15 not tested  2F 0.027more than 4 hours  2G 0.89 not tested  2H 0.124 not tested  2I 0.054more than 4 hours  2J 7.2 not tested  2K 0.29 similar to GLP-1  3A 0.007more than 4 hours  3B 0.007 more than 2 hours  3C 0.022 not tested  3D1.03 not tested  3E 0.06 more than 2 hours  3F 1.089 not tested  3G0.065 similar to GLP-1  5A 1.83 more than 4 hours  5B 0.53 more than 4hours  5C 1.056 not tested  5D 10.5 not tested  6A 0.144 similar toGLP-1  6B 0.52 not tested  6C 151.9 not tested  6D 0.282 not tested  6E0.60 more than 4 hours  8B 1.26 similar to GLP-1  8C 968.6 not tested 8D 106.2 not tested  9A 0.21 not tested  9B 0.03 more than 4 hours  9C0.05 more than 4 hours  9D 0.01 more than 4 hours  9E 133 not tested  9F0.07 more than 4 hours  9G 1.9 not tested  9H 0.1 not tested 11A 0.155not tested 11B 0.007 not tested 11C 0.011 not tested 12 0.028 not tested13 0.274 not tested 14J 0.054 not tested 14K 3.5 not tested 14L 0.05 nottested 14M 0.2 not tested (partial agonist) 14N 0.004 not tested 14O0.24 not tested 14P 0.25 not tested 14Q 0.02 not tested 14R 0.41 nottested (partial agonist) 15A 0.57 inactive 15B 1000 not tested 15C 2.46inactive 15D 1.7 inactive 16A 0.52 inactive 16B 0.18 similar to GLP-116C 1.1 inactive 16D 1 inactive 16E 1 inactive 16F 0.61 about 30 minutes16G 56 not tested 16H 1000 not tested 17A 105 not tested 17B 668 nottested 17C 10,000 not tested

Example 18 Preparation of Compounds in FIGS. 17A-F

A calculated 100 μmol of Rink amide resin was weighed into a reactionvessel in a Symphony peptide synthesizer, and the peptide elongation wascarried out following standard Fmoc peptide synthesis protocol up toresidue Pro³. The resulting peptide-resin intermediate (0.3 g, 0.0927mmol) was swollen in DCM with around 5% DMF and to the slurry was addedFmoc-Cys(Trt)-OH (0.351 g, 4 eq), followed byBromo-tris-pyrrolidino-phosphonium hexafluoro-phosphate (PyBrop) (0.34g, 4.8 eq) and 2,6-lutidine (0.27 mL, 15 eq). The vial was capped andheated in a microwave apparatus (Biotage Initiator8) at 100° C. for 11min. The resin was filtered and washed with DMF 6×, treated with 20%piperidine in DMF 2×25 min and washed with DMF 6×, DCM 4× followed bytreatment with 2% TFA/1.5% TIS in DCM 4×10 min. then washed with DCM 4×,DMF 2× and TMOF 3×. In a polypropylene syringe the resin is swollen inTMOF, followed by addition of compound 25 (0.2 g, 3 eq) and dry pyridine(0.06 mL, 5 eq). The resin was shaken at room temperature for 16 hours,followed by washings with TMOF 3×, DCM 3×, MeOH 3× and dried under highvacuum. Cleavage of the peptide from the resin was carried out with 10ml TFA/H₂O/PhOH/TIPS (95:2:2:1) (TFA is trifluoroacetic acid; TIPS istriisopropyl-silyl), precipitated by methyl-tert-Butyl ether. Theresidue was dissolved in 0.8 ml of MeOH and 0.8 ml of ACN. To thissolution 0.1 ml of DEA were added and the mixture stirred at roomtemperature overnight. The resulting residue was applied to areverse-phase high performance liquid chromatography (HPLC) column (C₁₈,20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford compound17A as a white powder (5.3 mg, 3%): Retention time in reverse phase-highperformance liquid chromatography (RP-HPLC) (C₁₈, 5-75% CH₃CN in 0.1%TFA/H₂O over 15 min) is 8.71 min; Calculated mass for C₁₄₆H₂₂₈N₃₈O₄₃S(M+H)⁺ 3235.74. found by liquid chromatography/mass spectrometry (LC-MS)1079.6 (M+3H)³⁺, 1619.9 (M+2H)²⁺.

The synthesis of compounds 17B and 17C was performed similar as reportedabove for compound 17A, with the difference of using Fmoc-D-Cys(Trt)-OHand Fmoc-Pen-OH, respectively. After purification compound 17B wasobtained as a white powder (8 mg, 7%): Retention time in reversephase-high performance liquid chromatography (RP-HPLC) (C₁₈, 5-75% CH₃CNin 0.1% TFA/H₂O over 15 min) is 8.55 min; Calculated mass forC₁₄₆H₂₂₈N₃₈O₄₃S (M+H)⁺ 3235.74. found by liquid chromatography/massspectrometry (LC-MS) 1079.6 (M+3H)³⁺, 1619.9 (M+2H)²⁺. Afterpurification compound 17C was obtained as a white powder (1.5 mg, 3%):Retention time in reverse phase-high performance liquid chromatography(RP-HPLC) (C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 8.55 min;Calculated mass for C₁₄₈H₂₃₂N₃₈O₄₃S (M+H)⁺ 3263.79. found by liquidchromatography/mass spectrometry (LC-MS) 1089.6 (M+3H)³⁺, 1632.9(M+2H)²⁺.

Example 19 Preparation of Compound 25

In a round bottom flask Fmoc-His(Boc)-OH (4 g, 1 eq) was dissolved inDCM (50 mL). To this solution was added DCC (1.9 g, 1.1 eq), EtSH (0.7mL, 1.1 eq) and DMAP (0.102 g, 0.1 eq). The mixture was stirred at roomtemperature for 6 h. The reaction mixture was filtered and the solutionconcentrated to yield 4.9 g of an off-white solid. The crude product waspurified by flash chromatography using Hex:AcOEt (1:1). Afterconcentration of the pure fractions the corresponding thioester wasobtained as a clear oil (3.6 g, 84%). The thioester (0.69 g, 1 eq) wasdissolved in THF (11 mL) and stirred under Argon atmosphere for fewminutes. To this solution was added Pd—C (0.180 g) and the mixturestirred under Argon for 10 min followed by dropwise addition of TES(0.75 mL, 3.5 eq) and the mixture stirred at room temperature. After 4 hone more equivalent of TES was added, one hour later the crude mixturewas filtered through a bed of silica/celite and washed with THF. Thefiltrate was concentrated to give a dark brown oil, which was purifiedby flash chromatography in a short (20 mL) silica gel column usingHex:AcOEt (2:8). After concentration of the pure fractions compound 25was obtained as a clear semisolid (0.26 g, 50%).

Example 20 Modified Exendin Peptides

N-Terminus conformationally constrained GLP-1 receptor agonist compoundsdescribed herein were covalently linked to one or more polyethyleneglycol and/or fatty acids, as described herein. In particular, thefollowing twelve compounds 23A-L were prepared:

Compound 23A:4-imidazopropionyl-dAla-PGTFTSDLSK¹²QMEEEAVRLFIE-WLKNGGPSSGAPPPS-NH₂,wherein K¹² was modified with—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂—(OCH₂CH₂)₂NH—C(═O)—(CH₂)₆—CH₃.

Compound 23B:4-imidazopropionyl-dAla-PGTFTSDLSK¹²QMEEEAVRLFIE-WLKNGGPSSGAPPPS-NH₂,wherein K¹² was modified with—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH(NH₂)—(CH₂)₇—CH₃.

Compound 23C:4-imidazopropionyl-dAla-PGTFTSDLSK¹²QMEEEAVRLFIE-WLKNGGPSSGAPPPS-NH₂,wherein K¹² was modified with—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH[NH—C(═O)(CH₂)₆CH₃]-(CH₂)₇—CH₃.

Compound 23D:4-imidazopropionyl-dAla-PGTFTSDLSK¹²QMEEEAVRLFIE-WLKNGGPSSGAPPPS-NH₂,wherein K¹² was modified with—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH₂CH₂—CH[C(OH)(═O)]—NH—C(═O)—(CH₂)₁₆-[C(OH)(═O)].

Compound 23E:4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEW-LK²⁷NGGPSSGAPPPS-NH₂,wherein K²⁷ was modified with—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—(CH₂)₆—CH₃.

Compound 23F:4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEW-LK²⁷NGGPSSGAPPPS-NH₂,wherein K²⁷ was modified with—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH(NH₂)—(CH₂)₇—CH₃.

Compound 23G:4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEW-LK²⁷NGGPSSGAPPPS-NH₂,wherein K²⁷ was modified with—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH[NH—C(═O)(CH₂)₆CH₃]—(CH₂)₇—CH₃.

Compound 23H:4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEW-LK²⁷NGGPSSGAPPPS-NH₂,wherein K²⁷ was modified with—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH₂CH₂—CH[C(OH)(═O)]—NH—C(═O)—(CH₂)₁₆-[C(OH)(═O)].

Compound 23I:4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEWLKN-GGPSSGAPPPS³⁹,wherein S³⁹ was modified with-Lys(NH₂)—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—(CH₂)₆—CH₃.

Compound 23J:4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEWLKN-GGPSSGAPPPS³⁹,wherein S³⁹ was modified with-Lys(NH₂)—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH(NH₂)—(CH₂)₇—CH₃.

Compound 23K:4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEWLKN-GGPSSGAPPPS³⁹,wherein S³⁹ was modified with-Lys(NH₂)—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH[NH—C(═O)(CH₂)₆CH₃]—(CH₂)₇—CH₃.

Compound 23L:4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEWLKN-GGPSSGAPPPS³⁹,wherein S³⁹ was modified with—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH₂CH₂—CH[C(OH)(═O)]—NH—C(═O)—(CH₂)₁₆—[C(OH)(═O)].

For each of Compound Nos. 23A-L above, a calculated 100 μmol ofFmoc-Rink-amide resin was weighed into a reaction vessel in a Symphonypeptide synthesizer, and peptide elongation was carried out followingstandard Fmoc peptide synthesis protocol. The dAla at position 2 wasincorporated manually in a polypropylene syringe. The orthogonalprotection (alloc group) of the side chain group (Lys¹², Lys²⁷ or Lys⁴⁰)was performed as follows: The peptide-resin was swollen in DCM anddimethylamino borane-complex (6 eq) followed after about 3 minutes bytetrakis(triphenylphosphine)palladium(0) (0.1 eq). The resin was shakenfor 15 minutes, washed with DCM 3× and the process repeated. Then, theresin was washed with DCM 3×, 10% DIEA in DCM 2×, DCM 3× and MeOH 2×. Atthis point, the peptide-resin gave a positive chloranil test. Theresulting peptide-resin intermediate (0.3 g, 0.0927 mmol) was swollen inDMF and to the slurry was added the corresponding polyethylene glycol(2.2 eq), followed by HBTU (0.035 g, 2.2 eq), HOBt (0.03 g, 2.2 eq) andNMM (0.04 mL, 4.4 eq). After 3 hours, the resin was washed with DMF 6×,treated with 20% piperidine in DMF 2×25 min and washed with DMF 6×. Theabove cycle was repeated with a second Fmoc-(polyethylene glycol)-OH inmost cases. The coupling of the corresponding fatty acid chain was doneusing two different microwave-assisted methods: the corresponding acylchloride (5 eq) and NMM (7 eq) were added to the resin swollen in a 1:1DMF:DCM mixture, and then heated using microwaves at 75° C. for 20 min(Biotage™, Initiator8); or the corresponding carboxylic or dicarboxylicacid (5 eq), HOAt (5 eq) and DIC (5 eq) were added to the slurry resinon DMF, and then heated using microwaves at 75° C. for 15 min (Biotage™,Initiator8). Cleavage of the peptide from the resin was done with 10 mLTFA/H₂O/PhOH/TIPS (95:2:2:1), then precipitated by methyl-tert-Butylether. The crude was dissolved and applied to a reverse-phase HPLCcolumn (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) toafford the titled compounds as white powders. All were characterized byLC-MS.

Each of these compounds was analyzed in an in vitro functional assay atthe GLP-1 receptor following the methods described in Example 16. Theresults are in Table 2:

TABLE 2 Cyclase GLP-1 Receptor Compound EC₅₀ (nM) 23A 0.027 23B 0.02723C 0.06 23D 0.22 23E 0.3 23F 0.1 23G 0.7 23H 0.08 23I 0.01 23J 0.01 23K0.02 23L 0.185

Example 21 Modified Exendin Peptides

N-Terminus conformationally constrained GLP-1 receptor agonist compoundsdescribed herein were covalently linked to one or more biotin, asdescribed herein. In particular, the following compounds 24A-L wereprepared:

Compound 24A: His-dAla-PGTFTSDLSKQMEEEAVRLFIEWL-Lys(biotin)-NGGPSSGAPPS-Lys[(NH₂)(biotin)]. Compound 24B:4-imidazopropionyl-GEGTFTSDLSKQMEEEAVRLFIEWL-Lys(biotin)-NGGPSSGAPPS-Lys[(NH₂)(biotin)]. Compound 24C:4-imidazopropionyl-GEGTFTSDLSKQMEEEAVRLFIEWLKN- Lys[(NH₂)(biotin)].Compound 24D: 4-imidazopropionyl-APGTFTSDLSKQMEEEAVRLFIEWLKN-Lys[(NH₂)(biotin)]. Compound 24E:His-dAla-PGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-Lys(NH₂)—(O—CH₂—CH₂)₂-KAKAEAEAKAKAEAEA-biotin Compound 24F:His-dAla-PGTFTSDLSKQMEEEAVRLFIEWLE[—(O—CH₂—CH₂)₂-(biotin)].-NGGPSSGAPPPS-NH₂. Compound 24G:His-dAla-PGTFTSDLSEK[—(O-CH₂-CH₂)₂-(biotin)]-QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂.

For each of Compound Nos. 24A-G above, a calculated 100 μmol ofFmoc-Rink-amide resin was weighed into a reaction vessel in a Symphonypeptide synthesizer, and peptide elongation was carried out followingstandard Fmoc peptide synthesis protocol. If an unnatural amino acid wasused at position 2 this was incorporated manually in a polypropylenesyringe. The orthogonal protection (alloc group) of the side chain group(Lys²⁷, Lys²⁸ or Lys⁴⁰) was performed as follows: The peptide-resin wasswollen in DCM and dimethylamino borane-complex (6 eq) followed afterabout 3 minutes by tetrakis(triphenylphosphine)palladium(0) (0.1 eq).The resin was shaken for 15 min, washed with DCM 3× and the processrepeated. Then, the resin was washed with DCM 3×, 10% DIEA in DCM 2×,DCM 3× and MeOH 2×. At this point, the peptide-resin gave a positivechloranil test. The biotin moiety (one or two) was coupled to the freeamino group using the standard solid-phase coupling conditions describedabove (HBTU, HOBt, NMM). Cleavage of the peptide from the resin was donewith 10 mL TFA/H₂O/PhOH/TIPS (95:2:2:1), then precipitated bymethyl-tert-Butyl ether. The crude was dissolved and applied to areverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 mingradient) to afford the titled compounds as white powders. All werecharacterized by LC-MS.

Each of these compounds was analyzed in an in vitro functional assay atthe GLP-1 receptor following the methods described in Example 16. Theresults are in Table 3:

TABLE 3 Cyclase GLP-1 Receptor Compound EC₅₀ (nM) 24A 0.04 24B 0.03 24C0.07 24D 0.77 24E 0.06 24F 0.06 24G 0.01

Example 22 Compounds of FIG. 10

With respect to FIG. 10, the following compounds 10J-10N were made:

10J: 4-imidazopropionyl-GPGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ 10K:4-imidazopropionyl-dAla-PGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ 10L:4-imidazopropionyl-Aib-PGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ 10M:4-imidazopropionyl-GEGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS-OH 10N:4-imidazopropionyl-dAla-PGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS-OH

For each of Compounds 10J-N, a calculated 100 μmol of Fmoc-Rink-amideresin was weighed into a reaction vessel in a Symphony peptidesynthesizer, and peptide elongation was carried out following standardFmoc peptide synthesis protocol. If an unnatural amino acid was used atposition 2 this was incorporated manually in a polypropylene syringe.Only for the compound in FIG. 10L, this procedure was carried out underthe following microwave-assisted conditions: a microwave vial was loadedwith the corresponding peptide-resin intermediate (0.3 g, 0.14 mmol),Fmoc-AA-OH (4 eq), PyBrop (0.32 g, 4.8 eq), 2,6-lutidine (0.25 mL, 15eq), dichloroethane (DCE) (2-3 mL) and DMF (˜0.3 mL). The vial wascapped and heated with microwaves (Biotage™, Initiator8) at 100° C. for11 min. The resin was filtered, washed with DCE and MeOH. Cleavage ofthe peptides from the resins was done with 10 mL TFA/H₂O/PhOH/TIPS(95:2:2:1), then precipitated by methyl-tert-Butyl ether. The crude wasdissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CNin 0.1% TFA/H₂O over 30 min gradient) to afford the titled compounds aswhite powders. All characterized by LC-MS.

Compounds 10J-L were analyzed in an in vitro functional assay at theGLP-1 receptor following the methods described in Example 16. Theresults are in Table 4:

TABLE 4 Cyclase GLP-1 Receptor Compound EC₅₀ (nM) 10J 0.1 10K 0.03 10L0.005 10M 0.007 10N 0.01

All publications and patent applications are incorporated herein byreference. Although the foregoing has been described in detail forpurposes of clarity of understanding, it will be apparent to one ofordinary skill in the art that changes and modifications may be madewithout departing from the spirit or scope of the disclosure or appendedclaims.

1-130. (canceled)
 131. A compound of Formula (B); Formula (C); orFormula (D): (SEQ ID NO: 1) (B)Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKN-Z; (SEQ ID NO: 2) (C)Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LK-R₁₀-Z; or (SEQ ID NO: 3)(D) Xaa₁Xaa_(2a)Xaa₃ Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKNGGPSSGAPPPS- Z;

wherein: Xaa₁ is a compound of Formula (1): Xaa₂ is Gly, dAla, Aib, Ala,Val, NMeAla, a compound of Formula (3), or a compound of Formula (4);and Xaa₂ is absent when Xaa₃ is:

Xaa₃ is Pro; Glu; Asp; a compound of Formula (2); a compound of Formula(3); a Compound of Formula (4);

Xaa₄ is Gly, dAla, or Aib; Xaa₁₄ is Leu or Met; Xaa₂₅ is Phe or Trp; R₁₀is QGGPSKEIIS (SEQ ID NO:22); NG; NGG; NGGP (SEQ ID NO:24); NGGPS (SEQID NO:25); NGGPSS (SEQ ID NO:26); NGGPSSG (SEQ ID NO:27); NGGPSSGA (SEQID NO:28); NGGPSSGAP (SEQ ID NO:29); NGGPSSGAPP (SEQ ID NO:30);NGGPSSGAPPP (SEQ ID NO:31); QGGPSSGAPPPS (SEQ ID NO:32); NGGPSSGAPPS(SEQ ID NO:33); NGGPSSGAPPSK (SEQ ID NO:34); NGGPSSGAPPS(K)₂₋₅ (SEQ IDNO:35); NGGPSSGAPPPSK (SEQ ID NO:36); or NK; and Z is OH or NH₂; whereinthe compound of Formula (1) is:

wherein R₂₀ and R₂₁ are each independently a single bond or a carbonatom; R₂₃, R₂₄, R₂₅ and R₂₆ are each independently absent, hydrogen,hydroxyl, C₁₋₆ alkyl, carboxyl, amino, or C₁₋₆ alkoxy;

is a single bond or a double bond; and R₂₁ is a chiral or achiral carbonatom; wherein the compound of Formula (2) is:

wherein Y₁ and Z₁ are each independently a single bond, a carbon, or asulfur; and W₁, W₂ and W₃ are each independently selected from hydrogen,C₁₋₆ alkyl, C₁₋₆ alkoxy, hydroxyl, and amino; and when one Y₁ or Z₁ issulfur, the sulfur may be bonded to two oxygen atoms to form a sulfonylgroup; and

is

or

; wherein the compound of Formula (3) is:

wherein Y₁ and Z₁ are each independently a single bond, a carbon, or asulfur; and W₁, W₂ and W₃ are each independently selected from hydrogen,C₁₋₆ alkyl, C₁₋₆ alkoxy, hydroxyl, and amino; and when one Y₁ or Z₁ issulfur, the sulfur may be bonded to two oxygen atoms to form a sulfonylgroup; and

is

or

; and wherein the compound of Formula (4) is:

wherein R₃₀, R₃₁, and R₃₂ are each independently hydrogen or a C₁₋₆alkyl; or R₃₀ and R₃₁, together with the nitrogen¹ and the carbon², forma 5-membered or 6-membered heterocyclic ring; or R₃₁ and R₃₂, togetherwith the carbon², form a 3-, 4-, or 5-membered carbocyclic ring. 132.The compound of claim 131, wherein the compound is a compound of Formula(B).
 133. The compound of claim 131, wherein the compound is a compoundof Formula (C).
 134. The compound of claim 131, wherein the compound isa compound of Formula (D).
 135. The compound of claim 131, wherein Xaa₃is Pro.
 136. The compound of claim 131, wherein the compound of Formula(B); Formula (C); and Formula (D) is selected from the group consistingof Pro³-exendin-4 (SEQ ID NO:40); Pro³,Leu¹⁴-exendin-4 (SEQ ID NO:41);Pro³,Leu¹⁴,Phe²⁵-exendin-4 (SEQ ID NO:42); Pro³-exendin-4(1-28) (SEQ IDNO:43); Pro³,Leu¹⁴-exendin-4(1-28) (SEQ ID NO:44);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-28) (SEQ ID NO:45); Pro³-exendin-4(1-29)(SEQ ID NO:51); Pro³,Leu¹⁴-exendin-4(1-29) (SEQ ID NO:75);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-29) (SEQ ID NO:52); Pro³-exendin-4(1-30)(SEQ ID NO:53); Pro³,Leu¹⁴-exendin-4(1-30) (SEQ ID NO:76);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-30) (SEQ ID NO:54); Pro³-exendin-4(1-31)(SEQ ID NO:55); Pro³,Leu¹⁴-exendin-4(1-31) (SEQ ID NO:77);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-31) (SEQ ID NO:56); Pro³-exendin-4(1-32)(SEQ ID NO:57); Pro³,Leu¹⁴-exendin-4(1-32) (SEQ ID NO:78);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-32) (SEQ ID NO:58); Pro³-exendin-4(1-33)(SEQ ID NO:59); Pro³,Leu¹⁴-exendin-4(1-33) (SEQ ID NO:79);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-33) (SEQ ID NO:60); Pro³-exendin-4(1-34)(SEQ ID NO:61); Pro³,Leu¹⁴-exendin-4(1-34) (SEQ ID NO:80);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-34) (SEQ ID NO:62); Pro³-exendin-4(1-35)(SEQ ID NO:63); Pro³,Leu¹⁴-exendin-4(1-35) (SEQ ID NO:81);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-35) (SEQ ID NO:64); Pro³-exendin-4(1-36)(SEQ ID NO:46); Pro³,Leu¹⁴-exendin-4(1-36) (SEQ ID NO:47);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-36) (SEQ ID NO:48); Pro³-exendin-4(1-37)(SEQ ID NO:65); Pro³,Leu¹⁴-exendin-4(1-37) (SEQ ID NO:82);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-37) (SEQ ID NO:66); Pro³-exendin-4(1-38)(SEQ ID NO:67); Pro³,Leu¹⁴-exendin-4(1-38) (SEQ ID NO:83);Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-38) (SEQ ID NO:68); Pro³-exendin-3 (SEQ IDNO:50); and HGPGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS (SEQ ID NO:49). 137.The compound of claim 131, wherein, for the compound of Formula (1), R₂₀and R₂₁ are each independently a single bond or a carbon atom; R₂₃ andR₂₄ are each independently absent, hydrogen, hydroxy, methyl, ethyl, orcarboxyl; R₂₅ and R₂₆ are each independently absent or hydrogen; ------is a single bond or a double bond; and R₂₁ is a chiral or achiral carbonatom.
 138. The compound of claim 131, wherein the compound of Formula(1) is a compound of Formula (1a), (1b), (1c), (1d), (1e), (1f), (1g),(1h), (1j), (1k), (1m), or (1n):


139. The compound of claim 131, wherein the compound of Formula (2) is acompound of Formula (2Z):

wherein R₄₀, R₄₁, and R₄₂ are each independently selected from the groupconsisting of hydrogen and C₁₋₄ alkyl.
 140. The compound of claim 131,wherein the compound of Formula (2) is a compound of Formula (2a), (2b),(2c), (2d), (2e), (2f), (2g), (2h), or (2j):


141. The compound of claim 131, wherein the compound of Formula (3) is acompound of Formula (3a), (3b), or (3c):


142. The compound of claim 131, wherein, for the compound of Formula(4), R₃₀, R₃₁, and R₃₂ are each independently hydrogen, methyl, orethyl; or R₃₀ and R₃₁, together with the nitrogen¹ and carbon², form a5-membered or 6-membered heterocyclic ring; or R₃₁ and R₃₂, togetherwith the carbon², form a 3-, or 4-membered carbocyclic ring.
 143. Thecompound of claim 131, wherein the compound of Formula (4) is a compoundof Formula (4a), (4b), (4c), (4d), or (4e):


144. A compound of Formula (A): Xaa₁Xaa₂Xaa₃Xaa₄-Z; wherein: Xaa₁ is acompound of Formula (1); Xaa₂ is Gly, dAla, Aib, Ala, Val, NMeAla, acompound of Formula (3), or a compound of Formula (4); and Xaa₂ isabsent when Xaa₃ is:

Xaa₃ is Pro; a compound of Formula (2); a compound of Formula (3); aCompound of Formula (4);

Xaa₄ is Gly, dAla, or Aib; and Z is OH or NH₂; wherein the compound ofFormula (1) is:

wherein R₂₀ and R₂₁ are each independently a single bond or a carbonatom; R₂₃, R₂₄, R₂₅ and R₂₆ are each independently absent, hydrogen,hydroxyl, C₁₋₆ alkyl, carboxyl, or C₁₋₄ alkoxy;

is a single bond or a double bond; and R₂₁ is a chiral or achiral carbonatom; wherein the compound of Formula (2) is:

wherein Y₁ and Z₁ are each independently a single bond, a carbon, or asulfur; and W₁, W₂ and W₃ are each independently selected from hydrogen,C₁₋₆ alkyl, C₁₋₆ alkoxy, hydroxyl, and amino; and when one Y₁ or Z₁ issulfur, the sulfur may be bonded to two oxygen atoms to form a sulfonylgroup; and

is

or

; wherein the compound of Formula (3) is:

wherein Y₁ and Z₁ are each independently a single bond, a carbon, or asulfur; and W₁, W₂ and W₃ are each independently selected from hydrogen,C₁₋₆ alkyl, C₁₋₆ alkoxy, hydroxyl, and amino; and when one Y₁ or Z₁ issulfur, the sulfur may be bonded to two oxygen atoms to form a sulfonylgroup; and

is

or

; and wherein the compound of Formula (4) is:

wherein R₃₀, R₃₁, and R₃₂ are each independently hydrogen or a C₁₋₆alkyl; or R₃₀ and R₃₁, together with the nitrogen¹ and the carbon², forma 5-membered or 6-membered heterocyclic ring; or R₃₁ and R₃₂, togetherwith the carbon², form a 3-, 4-, or 5-membered carbocyclic ring. 145.The compound of claim 144, wherein Xaa₃ is Pro.
 146. The compound ofclaim 144, wherein Xaa₁ is


147. A compound of Formula (E):Xaa₁Xaa₂Xaa₃Xaa₄TFTSDVSSYXaa₁₄EGQAAKEFIAXaa₂₅LVXaa₂₈GRXaa₃₁-Z; wherein:Xaa₁ is a compound of Formula (1); Xaa₂ is dAla, Aib, Val, NMeAla, acompound of Formula (3), or a compound of Formula (4); and Xaa₂ isabsent when Xaa₃ is:

Xaa₃ is Pro; Glu; Asp; a compound of Formula (2); a compound of Formula(3); a Compound of Formula (4);

Xaa₄ is Gly, dAla, or Aib; Xaa₁₄ is Leu or Met; Xaa₂₅ is Phe or Trp;Xaa₂₈ is Lys or Arg; Xaa₃₁ is Gly or absent; and Z is OH or NH₂, whereinthe compound of Formula (1) is:

wherein R₂₀ and R₂₁ are each independently a single bond or a carbonatom; R₂₃, R₂₄, R₂₅ and R₂₆ are each independently absent, hydrogen,hydroxyl, C₁₋₆ alkyl, carboxyl, or C₁₋₆ alkoxy;

is a single bond or a double bond; and R₂₁ is a chiral or achiral carbonatom; wherein the compound of Formula (2) is:

wherein Y₁ and Z₁ are each independently a single bond, a carbon, or asulfur; and W₁, W₂ and W₃ are each independently selected from hydrogen,C₁₋₆ alkyl, C₁₋₆ alkoxy, hydroxyl, and amino; and when one Y₁ or Z₁ issulfur, the sulfur may be bonded to two oxygen atoms to form a sulfonylgroup; and

is

or

; wherein the compound of Formula (3) is:

wherein Y₁ and Z₁ are each independently a single bond, a carbon, or asulfur; and W₁, W₂ and W₃ are each independently selected from hydrogen,C₁₋₆ alkyl, C₁₋₆ alkoxy, hydroxyl, and amino; and when one Y₁ or Z₁ issulfur, the sulfur may be bonded to two oxygen atoms to form a sulfonylgroup; and

is

or

; and wherein the compound of Formula (4) is:

wherein R₃₀, R₃₁, and R₃₂ are each independently hydrogen or a C₁₋₆alkyl; or R₃₀ and R₃₁, together with the nitrogen¹ and the carbon², forma 5-membered or 6-membered heterocyclic ring; or R₃₁, and R₃₂, togetherwith the carbon², form a 3-, 4-, or 5-membered carbocyclic ring. 148.The compound of claim 147, wherein Xaa₃ is Pro.
 149. A compound ofFormula (F): Xaa₁Xaa₂Xaa₃-Z, wherein: Xaa₁ is a compound of Formula (1):

wherein R₂₀ and R₂₁ are each independently a single bond or a carbonatom; R₂₃, R₂₄, R₂₅ and R₂₆ are each independently absent, hydrogen,hydroxyl, C₁₋₆ alkyl, carboxyl, amino, or C₁₋₆ alkoxy;

is a single bond or a double bond; and R₂₁ is a chiral or achiral carbonatom; Xaa₂ is Gly, Ala, dAla, or Aib; Xaa₃ is:

wherein * indicates a chiral carbon atom; and R₂ is hydrogen or a C₁₋₄alkyl; and Z is (i) OH; (ii) NH₂; (iii)TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKN-Z₁ (SEQ ID NO:18) (iv)TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKNGGPSSGAPPPS-Z₁ (SEQ ID NO:19) (v)TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LK-R₁₀-Z₁ (SEQ ID NO:20) or (vi)TFTSDVSSYXaa₁₄EGQAAKEFIAXaa₂₅LVXaa₂₈GRXaa₃₁-Z₁ (SEQ ID NO:21); wherein:Z₁ is OH or NH₂; Xaa₁₄ is Leu or Met; Xaa₂₅ is Phe or Trp; Xaa₂₈ is Lysor Arg; Xaa₃₁ is Gly or absent; and R₁₀ is QGGPSKEIIS (SEQ ID NO:22);NG; NGG; NGGP (SEQ ID NO:24); NGGPS (SEQ ID NO:25); NGGPSS (SEQ IDNO:26); NGGPSSG (SEQ ID NO:27); NGGPSSGA (SEQ ID NO:28); NGGPSSGAP (SEQID NO:29); NGGPSSGAPP (SEQ ID NO:30); NGGPSSGAPPP (SEQ ID NO:31);NGGPSSGAPPS (SEQ ID NO:32); NGGPSSGAPPSK (SEQ ID NO:33);NGGPSSGAPPS(K)₂₋₆ (SEQ ID NO:34); NGGPSSGAPPPSK (SEQ ID NO:35); or NK.150. A method for treating diabetes, treating insulin resistance,treating postprandial hyperglycemia, lowering blood glucose levels,lowering HbA1c levels, stimulating insulin release, reducing gastricmotility, delaying gastric emptying, reducing food intake, reducingappetite, reducing weight, treating overweight, or treating obesity in apatient in need thereof comprising administering to the patient atherapeutically effective amount of the compound of claim 1 to treatdiabetes, treat insulin resistance, treat postprandial hyperglycemia,lower blood glucose levels, to lower HbA1c levels, stimulate insulinrelease, reduce gastric motility, delay gastric emptying, reduce foodintake, reduce appetite, reduce weight, treat overweight, or treatobesity in the patient.